JQW, SZ, SD and KC are supported by grant from the National Institutes of Health and the Staman Ogilvie Fund&#8212;Memorial Hermann Foundation.

1. EML Cell Culture and Separation of Lin-CD34+ and Lin-CD34- Cells Using Magnetic Cell Sorting System and Fluorescence-activated Cell Sorting Method
<ol>
	<li>Preparation of baby hamster kidney (BHK) cell culture medium for stem cell factor collection:
	<ol>
		<li>Culture BHK cells in DMEM medium containing 10% FBS in 25 cm2 flask (Table 1) at 37 &#176;C, 5% CO2 in a cell culture incubator.</li>
		<li>When cells grow to 80 - 90% confluence, wash cells once with 10 ml of PBS. Add 5 ml of 0.25% trypsin-EDTA solution to the monolayer and incubate the cells for 1-5 min at room temperature (RT) until the cells are detached.</li>
		<li>Pipet the solution up and down gently to break up clumps of cells. Add 5 ml of complete DMEM to the flask to stop trypsin activity. Collect cells by centrifugation at 200 x g for 5 min at RT.</li>
		<li>Remove the medium and resuspend the cell pellet in 10 ml of fresh BHK cell culture medium.</li>
		<li>Transfer 2 ml of the cell suspension from the step 1.1.4 to a new 75 cm2 flask and add 48 ml of fresh BHK cell culture medium to the flask.</li>
		<li>Culture the BHK cells for two days and collect the culture medium. Passage the medium through a 0.45 &#956;m filter. Store the medium in -20 &#176;C until further use.</li>
	</ol>
	</li>
	<li>EML cell culture:
	<ol>
		<li>Culture EML cells (in suspension) in EML basic medium containing BHK cell culture medium (Table 1) at 37 &#176;C, 5% CO2 in a cell culture incubator.</li>
		<li>Maintain the EML cells at low cell density (0.5-5 x 105 cells/ml) with the peak density less than 6 x 105 cells/ml. Split the cells every 2-3 days at the ratio of 1:5. Passage EML cells gently and discard the culture after passaging for 10 generations.</li>
	</ol>
	</li>
	<li>Depletion of lineage positive cells:
	<ol>
		<li>Harvest the EML cells by centrifugation at 200 x g for 5 min and wash the cells once with PBS. Collect the cells by centrifugation at 200 x g for 5 min.</li>
		<li>Resuspend the cells with PBS and count the cells with a hemocytometer. Determine the antibody concentration in the subsequent cell separation step according to the number of the cells (please refer to the instructions offered by the provider of the cell isolation system).</li>
		<li>Isolate the lineage negative (Lin-) cells using lineage antibody cocktail (cocktail of biotin-conjugated monoclonal antibodies CD5, CD45R (B220), CD11b, Anti-Gr-1(Ly-6G/C), 7-4 and Ter-119) and a magnetic activated cell sorting system according to manufacturer&#8217;s instructions.</li>
	</ol>
	</li>
	<li>Separation of Lin-CD34+ and Lin-CD34- cells:
	<ol>
		<li>Spin down the Lin- cells from the step 1.3.3 at 200 x g for 5 min. Resuspend the cell pellet with PBS and count the cells with a hemocytometer.</li>
		<li>Wash the cells twice with FACS buffer and pellet the cells at 200 x g for 5 min.</li>
		<li>Label five 1.5 ml microcentrifuge tubes with the number 1, 2, 3, 4, 5 respectively. Resuspend the cells with 100 &#181;l FACS buffer per 106 cells (106 cells per tube).</li>
		<li>Add 1 &#181;g of Anti-Mouse CD34 FITC antibody to tube 1 and tube 2 and mix the tubes gently.</li>
		<li>Incubate all tubes at 4 &#176;C for 1 hr in the dark.</li>
		<li>Add 0.25 &#181;g of PE-conjugated Anti-Sca1 antibody and 20 &#181;l of APC-conjugated Lineage Cocktail antibodies to tube 1, 0.25 &#181;g of PE-conjugated Anti-Sca1 antibody to tube 3, and 20 &#181;l of APC-conjugated Lineage Cocktail antibodies to tube 4.</li>
		<li>Mix all the tubes gently and incubate the cells at 4 &#176;C for an additional 30 min in the dark.</li>
		<li>Add 300 &#181;l of FACS buffer to the cells and spin down the cells at 200 x g for 5 min.</li>
		<li>Wash the cells with 500 &#181;l of FACS buffer for three times.</li>
		<li>Resuspend the cell pellet in 500 &#181;l of FACS buffer.</li>
		<li>Use the cells in tubes 2, 3, 4, and 5 for setting up compensation. Isolate Lin-SCA+CD34+ and Lin-SCA-CD34- cells in tube 1 using FACS Aria.</li>
	</ol>
	</li>
</ol>
2. RNA Preparation and Library Construction for High-throughput Sequencing
<ol>
	<li>Isolation, quality analysis and quantification of RNA:
	<ol>
		<li>Extract total RNA from Lin-CD34+ and Lin-CD34- cells respectively using TRIzol following the manufactures&#8217; protocol.</li>
		<li>Remove the contaminated DNA using deoxyribonuclease I (DNase I) following the manufacture&#8217;s protocol. Optionally, store the RNA at -80 &#176;C at this step for further use.</li>
		<li>Assess the quality of total RNA using Bioanalyzer according to the instructions offered by the supplier. Use RNA sample with RNA Integrity Number (RIN) lager than 9.</li>
	</ol>
	</li>
	<li>Library Construction and high-throughput sequencing: 
	NOTE: This protocol describes RNA-Seq using Illumina platform. For other sequencing platforms, different library preparation methods are required.
	<ol>
		<li>Use 0.1-4 &#181;g of high quality total RNA per sample for library preparation. Normally 2 &#181;g of&#160;total RNA can be extracted from 105 EML cells.</li>
		<li>Use a RNA-sequencing sample preparation system for&#160;RNA purification and fragmentation, first and second strand cDNA synthesis, end repair, 3&#8217; ends adenylation, adapter ligation and PCR amplification, following the detailed standard procedures from the provider&#8217;s instructions.
		<ol>
			<li>Positively select PolyA mRNA&#160;using oligo-dT magnetic beads and fragment the mRNA.</li>
			<li>Perform reverse transcription using random primers to obtain the cDNA and subsequently synthesize the second strand of cDNA to generate double stranded cDNA.</li>
			<li>Remove the 3&#39; overhangs and fill the 5&#39; overhangs by DNA polymerase. Adenylate 3&#39; ends to prevent cDNA fragments from ligating to one another.</li>
			<li>Add multiplex indexing adapters to both ends of the dscDNA. Perform PCR for the enrichment of DNA fragments.</li>
		</ol>
		</li>
		<li>Measure the A260/A280 to obtain information about the concentration of library using a spectrophotometer.</li>
		<li>Assess the library quality and measure the size range of DNA fragments using a Bioanalyzer.</li>
	</ol>
	</li>
</ol>
3. Data Analysis
For reference of software used in this part, please see (Table 2).
<ol>
	<li>Data file processing for downstream analysis:
	<ol>
		<li>Convert .bcl (base call file) file to .fastq file using CASAVA software (Illumina, version 1.8.2).
		<ol>
			<li>Fire up the &#8216;Terminal&#8217; in Linux system. Go to the data folder that contains the data file from an Illumina HiSeq2000 sequencing machine. Suppose the result folder is &#8216;NASboy1/JiaqianLabData/HiSeq_RUN/2013_07_11/130627_SN860_0309_A_2013-166_H0PW9ADXX/&#8217;, type in the command in Figure S1A, and enter the data folder.</li>
			<li>Install CASAVA 1.8.2 in the Linux system. Suppose the outputfolder is &#8216;Unaligned&#8217;, use the command in Figure S1B to prepare the configuration file for converting. Use the option --fastq-cluster-count 0 to ensure only one .fastq file is created for each sample. The generated .fastq file is in .gz format. Unzip it for downstream analysis (Figure S1B).</li>
			<li>After the &#8216;Unaligned&#8217; folder has been generated, go to the &#8216;Unaligned&#8217; folder (Figure S1C).</li>
			<li>Use the command in Figure S1D to begin the converting process. The &#8216;-j&#8217; parameter supplies the cpu number that will be used.</li>
			<li>After the system finished the converting process, go to the result folder under &#8216;Unaligned&#8217; folder (Figure S1E).</li>
			<li>Use the command in Figure S1F to decompress the .fastq.gz file into .fastq file under each sample folder.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Detect novel transcripts and evaluate the expression level using Tuxedo Suite26:
	<ol>
		<li>Map the paired-end RNA-Seq reads to the mouse reference genome (UCSC version mm9, obtained from <a href="http://cufflinks.cbcb.umd.edu/igenomes.html" target="_blank">http://cufflinks.cbcb.umd.edu/igenomes.html</a>) using Tophat software (version 1.3.3)27, which uses the Bowtie read mapper (version 0.12.7)28. Tophat is supplied with &#8220;-no-novel-juncs&#8221; option to improve estimation accuracy of expression level.
		<ol>
			<li>Put the .fastq files in a folder where the mapping process will be implemented. Suppose there are 2 .fastq files (rename to Example1.read1, Example1.read2) for a paired-end sequencing sample, use the command in Figure S2 to do the mapping (adjust the parameters according to the system setting). The &#8220;-p&#8221; parameter supplies the cpu number that will be used. The &#8220;&#8211;r&#8221; and &#8220;&#8211;mate-std-dev&#8221; parameters can be obtained from library QC or inferred from a subset of aligned reads (Figure S2).</li>
		</ol>
		</li>
		<li>Assemble the mapped reads into RNA transcripts using the Cufflinks software (version 1.3.0)29. Run Cufflinks using the annotation file of known genes (same .gtf file used by Tophat) and .bam file produced by Tophat.
		<ol>
			<li>After Tophat finished running, in the same folder, use the command in Figure S3A to run cufflinks to construct transcriptome and estimate transcript expression level. The &#8216;mm9_repeatMasker.gtf&#8217; and genome sequence files in the &#8216;GenomeSeqMM9&#8217; folder can be obtained from UCSC Genome Browser.</li>
			<li>The resulting genes.expr and transcripts.expr files contain the expression value of genes and transcripts (isoforms). Copy and paste the file contents to an Excel file and manipulate with spreadsheet application (Figure S3B).</li>
			<li>Use the command in Figure S3C to compare the resulting &#8216;transcripts.gtf&#8217; file to the reference &#8216;mm9_genes.gtf&#8217; file in order to identify novel transcripts.</li>
			<li>The resulting .tmap file contains the comparison result. Copy and paste the file contents to an Excel file and manipulate with spreadsheet application. Transcripts with class code &#8216;u&#8217; can be considered as &#8216;novel&#8217; compared to the reference .gtf file provided (Figure S3D). 
			NOTE: For downstream analysis convenience, set the FPKM values to 0.1 if the values are under 0.1. 
			NOTE: Step 3.2.3 - 3.2.6 is optional for those who wish to improve accuracy of novel transcripts&#8217; expression estimation. This will take a much longer time, because mapping and transcriptome construction need to be run more than once.</li>
		</ol>
		</li>
		<li>Run Tophat using default parameters and then run cufflinks to generated .gtf file using the command in Figure S3E.</li>
		<li>Compare the resulting .gtf file to the reference genome .gtf file using the command in Figure S3F.</li>
		<li>Parse the resulted .tmap file as described in the step 3.2.2.4. Copy and paste the file contents to an Excel file and manipulate with spreadsheet application. Transcripts with class code &#8216;u&#8217; can be considered as &#8216;novel&#8217; compared to the reference .gtf file provided.</li>
		<li>After the step 3.2.5, there is a .combined.gtf file in the folder which can be used as the reference .gtf file. A second run of Tophat and cufflinks can be performed as described in the step 3.2.1 and 3.2.2 to obtain a more accurate FPKM estimation of novel transcripts.</li>
	</ol>
	</li>
	<li>Detect differentially expressed genes using DESeq package30.
	<ol>
		<li>The input of DESeq is a raw read counts table. To obtain such a table, use the htseq-count script distributed with the HTSeq Python package which can be downloaded from HTSeq website (<a href="http://www-huber.embl.de/users/anders/HTSeq/doc/count.html" target="_blank">http://www-huber.embl.de/users/anders/HTSeq/doc/count.html</a>).
		<ol>
			<li>Ensure that samtools, python, and htseq-count programsare installed in the system. Obtain raw read count numbers from tophat output by using the command in Figure S4A.</li>
			<li>Prepare &#8216;Raw_Count_Table.txt&#8217;, &#8216;ExperimentDesign.txt&#8217; files using Excel. Copy and save the content in .txt format for the DESeq R package (Figure S4B).</li>
			<li>Install R program in the system. In the terminal, type &#8216;R&#8217; and press ENTER.A screen message will appearas showed in Figure S4C.</li>
			<li>Read &#8216;Raw_Count_Table.txt&#8217;, &#8216;ExperimentDesign.txt&#8217; into R using the command in Figure S4D.</li>
			<li>Load DESeq package using the command in Figure S4E.</li>
			<li>Factorize conditions in R (Figure S4F).</li>
			<li>Use the command in Figure S4G to run negative binominal test on the normalized count table.</li>
			<li>Use the command in Figure S4H to output significant differential expressed genes in a .csv file.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Lookup transcription factors&#8217; (TFs) FPKM values across samples using Excel. Intersect DE gene table and TFs table. Genes belong to both table are differentially expressed transcription factors.
	<ol>
		<li>Go to the website <a href="http://www.bioguo.org/AnimalTFDB/download.php" target="_blank">http://www.bioguo.org/AnimalTFDB/download.php</a> and download the transcription factors. Then lookup the DE transcription factors in the Excel (Figure S5).</li>
	</ol>
	</li>
	<li>Generating .bigwig file for UCSC genome browser visualization.
	<ol>
		<li>Download &#8216;bedtools&#8217; software package from the website <a href="https://github.com/arq5x/bedtools2" target="_blank">https://github.com/arq5x/bedtools2</a> and install the software in the system31. Download the UCSC tools &#8216;bedGraphToBigWig&#8217; from the website <a href="http://hgdownload.cse.ucsc.edu/admin/exe/" target="_blank">http://hgdownload.cse.ucsc.edu/admin/exe/</a> and install the software in the system.</li>
		<li>In the folder containing the .bam file, use the command in Figure S6A to convert .bam file generated by tophat into .bed file.</li>
		<li>After the .bed file is produced, use the command in Figure S6B to generate .bigwig file. The file &#8216;ChromInfo.txt&#8217; can be obtained from following url: <a href="http://hgdownload.cse.ucsc.edu/goldenPath/mm9/database/chromInfo.txt.gz" target="_blank">http://hgdownload.cse.ucsc.edu/goldenPath/mm9/database/chromInfo.txt.gz</a>.</li>
		<li>Observe a custom track on UCSC Genome Browser. Refer to the website <a href="http://genome.ucsc.edu/goldenPath/help/customTrack.html" target="_blank">http://genome.ucsc.edu/goldenPath/help/customTrack.html</a> on how to display a custom track using UCSC genome browser.</li>
	</ol>
	</li>
</ol>
<img alt="Figure S1" fo:content-width="5in" src="/files/ftp_upload/52104/52104supplementalfig1.jpg" /> 
Figure S1: Converting .bcl file to .fastq file using CASAVA software.
<img alt="Figure S2" fo:content-width="5in" src="/files/ftp_upload/52104/52104supplementalfig2.jpg" /> 
Figure S2: Mapping reads to reference genome using Tophat.
<img alt="Figure S3" fo:content-width="5in" src="/files/ftp_upload/52104/52104supplementalfig3.jpg" /> 
Figure S3: Detection of novel transcripts and expression level estimation.
<img alt="Figure S4" fo:content-width="3.4in" src="/files/ftp_upload/52104/52104supplementalfig4.jpg" /> 
Figure S4: Calling differential expressed gene using DESeq package.
<img alt="Figure S5" fo:content-width="5in" src="/files/ftp_upload/52104/52104supplementalfig5.jpg" /> 
Figure S5: Identification of differentially expressed transcription factors.
<img alt="Figure S6" fo:content-width="5in" src="/files/ftp_upload/52104/52104supplementalfig6.jpg" /> 
Figure S6: Converting mapping result for data visualization.

<ol>
	<li>Chambers, S. M., Goodell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell+aging:+wrinkles+in+stem+cell+potential.">Hematopoietic stem cell aging: wrinkles in stem cell potential.</a> Stem Cell Rev. 3, 201-211 (2007).</li><li>Wu, J. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tcf7+is+an+important+regulator+of+the+switch+of+self-renewal+and+differentiation+in+a+multipotential+hematopoietic+cell+line.">Tcf7 is an important regulator of the switch of self-renewal and differentiation in a multipotential hematopoietic cell line.</a> PLoS genetics. 8, (2012).</li><li>Ye, Z. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+interactions+in+EML+cell+stimulation+by+stem+cell+factor+and+IL-3.">Complex interactions in EML cell stimulation by stem cell factor and IL-3.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, 4882-4887 (2011).</li><li>Tsai, S., Bartelmez, S., Sitnicka, E., Collins, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphohematopoietic+progenitors+immortalized+by+a+retroviral+vector+harboring+a+dominant-negative+retinoic+acid+receptor+can+recapitulate+lymphoid,+myeloid,+and+erythroid+development.">Lymphohematopoietic progenitors immortalized by a retroviral vector harboring a dominant-negative retinoic acid receptor can recapitulate lymphoid, myeloid, and erythroid development.</a> Genes Dev. 8, 2831-2841 (1994).</li><li>Weiler, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=D3:+a+gene+induced+during+myeloid+cell+differentiation+of+Linlo+c-Kit++Sca-1(+)+progenitor+cells.">D3: a gene induced during myeloid cell differentiation of Linlo c-Kit+ Sca-1(+) progenitor cells.</a> Blood. 93, 527-536 (1999).</li><li>Ye, Z. J., Kluger, Y., Lian, Z., Weissman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+types+of+precursor+cells+in+a+multipotential+hematopoietic+cell+line.">Two types of precursor cells in a multipotential hematopoietic cell line.</a> Proc Natl Acad Sci U S A. 102, 18461-18466 (2005).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nature reviews. Genetics. 10, 57-63 (2009).</li><li>Chu, Y., Corey, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+sequencing:+platform+selection,+experimental+design,+and+data+interpretation.">RNA sequencing: platform selection, experimental design, and data interpretation.</a> Nucleic acid therapeutics. 22, 271-274 (2012).</li><li>Hornett, E. A., Wheat, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+RNA-Seq+analysis+in+non-model+species:+assessing+transcriptome+assemblies+as+a+scaffold+and+the+utility+of+evolutionary+divergent+genomic+reference+species.">Quantitative RNA-Seq analysis in non-model species: assessing transcriptome assemblies as a scaffold and the utility of evolutionary divergent genomic reference species.</a> BMC genomics. 13, 361 (2012).</li><li>Eswaran, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+sequencing+of+cancer+reveals+novel+splicing+alterations.">RNA sequencing of cancer reveals novel splicing alterations.</a> Scientific reports. 3, 1689 (2013).</li><li>Wang, E. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+isoform+regulation+in+human+tissue+transcriptomes.">Alternative isoform regulation in human tissue transcriptomes.</a> Nature. 456, 470-476 (2008).</li><li>Wu, J. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+transcriptomes+during+neural+differentiation+of+human+embryonic+stem+cells+revealed+by+short,+long,+and+paired-end+sequencing.">Dynamic transcriptomes during neural differentiation of human embryonic stem cells revealed by short, long, and paired-end sequencing.</a> Proceedings of the National Academy of Sciences of the United States of America. 107, 5254-5259 (2010).</li><li>Loraine, A. E., McCormick, S., Estrada, A., Patel, K., Qin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq+of+Arabidopsis+pollen+uncovers+novel+transcription+and+alternative+splicing.">RNA-seq of Arabidopsis pollen uncovers novel transcription and alternative splicing.</a> Plant physiology. 162, 1092-1109 (2013).</li><li>Edgren, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+fusion+genes+in+breast+cancer+by+paired-end+RNA-sequencing.">Identification of fusion genes in breast cancer by paired-end RNA-sequencing.</a> Genome biology. 12, 6 (2011).</li><li>Ilott, N. E., Ponting, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+long+non-coding+RNAs+using+RNA+sequencing.">Predicting long non-coding RNAs using RNA sequencing.</a> Methods. 63, 50-59 (2013).</li><li>Sun, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+novel+long+non-coding+RNAs+based+on+RNA-Seq+data+of+mouse+Klf1+knockout+study.">Prediction of novel long non-coding RNAs based on RNA-Seq data of mouse Klf1 knockout study.</a> BMC bioinformatics. 13, 331 (2012).</li><li>Luo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+expression+analysis+using+the+Illumina+microRNA-Seq+Platform.">MicroRNA expression analysis using the Illumina microRNA-Seq Platform.</a> Methods in molecular biology. 822, 183-188 (2012).</li><li>Bolduc, F., Hoareau, C., St-Pierre, P., Perreault, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-depth+sequencing+of+the+siRNAs+associated+with+peach+latent+mosaic+viroid+infection.">In-depth sequencing of the siRNAs associated with peach latent mosaic viroid infection.</a> BMC molecular biology. 11, 16 (2010).</li><li>Chepelev, I., Wei, G., Tang, Q., Zhao, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+single+nucleotide+variations+in+expressed+exons+of+the+human+genome+using+RNA-Seq.">Detection of single nucleotide variations in expressed exons of the human genome using RNA-Seq.</a> Nucleic acids research. 37, 106 (2009).</li><li>Djari, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-based+single+nucleotide+polymorphism+discovery+in+bovine+muscle+using+next-generation+transcriptomic+sequencing.">Gene-based single nucleotide polymorphism discovery in bovine muscle using next-generation transcriptomic sequencing.</a> BMC genomics. 14, 307 (2013).</li><li>Murphy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+studies+using+microarrays:+principles,+problems,+and+prospects.">Gene expression studies using microarrays: principles, problems, and prospects.</a> Advances in physiology education. 26, 256-270 (2002).</li><li>Chen, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq+characterization+of+spinal+cord+injury+transcriptome+in+acute/subacute+phases:+a+resource+for+understanding+the+pathology+at+the+systems+level.">RNA-seq characterization of spinal cord injury transcriptome in acute/subacute phases: a resource for understanding the pathology at the systems level.</a> PLoS one. 8, 72567 (2013).</li><li>Marioni, J. C., Mason, C. E., Mane, S. M., Stephens, M., Gilad, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq:+an+assessment+of+technical+reproducibility+and+comparison+with+gene+expression+arrays.">RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays.</a> Genome research. 18, 1509-1517 (2008).</li><li>Ramskold, D., Kavak, E., Sandberg, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+analyze+gene+expression+using+RNA-sequencing+data.">How to analyze gene expression using RNA-sequencing data.</a> Methods in molecular biology. 802, 259-274 (2012).</li><li>Glenn, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+guide+to+next-generation+DNA+sequencers.">Field guide to next-generation DNA sequencers.</a> Mol Ecol Resour. 11, 759-769 (2011).</li><li>Trapnell, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+gene+and+transcript+expression+analysis+of+RNA-seq+experiments+with+TopHat+and+Cufflinks.">Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks.</a> Nature protocols. 7, 562-578 (2012).</li><li>Trapnell, C., Pachter, L., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TopHat:+discovering+splice+junctions+with+RNA-Seq.">TopHat: discovering splice junctions with RNA-Seq.</a> Bioinformatics. 25, 1105-1111 (2009).</li><li>Langmead, B., Trapnell, C., Pop, M., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+and+memory-efficient+alignment+of+short+DNA+sequences+to+the+human+genome.">Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.</a> Genome biology. 10, 25 (2009).</li><li>Trapnell, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcript+assembly+and+quantification+by+RNA-Seq+reveals+unannotated+transcripts+and+isoform+switching+during+cell+differentiation.">Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation.</a> Nature. 28, 511-515 (2010).</li><li>Anders, S., Huber, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+analysis+for+sequence+count+data.">Differential expression analysis for sequence count data.</a> Genome biology. 11, 106 (2010).</li><li>Quinlan, A. R., Hall, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BEDTools:+a+flexible+suite+of+utilities+for+comparing+genomic+features.">BEDTools: a flexible suite of utilities for comparing genomic features.</a> Bioinformatics. 26, 841-842 (2010).</li><li>Cheranova, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq+analysis+of+transcriptomes+in+thrombin-treated+and+control+human+pulmonary+microvascular+endothelial+cells.">RNA-seq analysis of transcriptomes in thrombin-treated and control human pulmonary microvascular endothelial cells.</a> J Vis Exp. , (2013).</li><li>Zhang, H. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AnimalTFDB:+a+comprehensive+animal+transcription+factor+database.">AnimalTFDB: a comprehensive animal transcription factor database.</a> Nucleic acids research. 40, 144-149 (2012).</li><li>Wu, J. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+analysis+of+transcribed+loci+in+ENCODE+regions+using+RACE+sequencing+reveals+extensive+transcription+in+the+human+genome.">Systematic analysis of transcribed loci in ENCODE regions using RACE sequencing reveals extensive transcription in the human genome.</a> Genome Biol. 9, 3 (2008).</li><li>Wu, J. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+RT-PCR+recovery+of+full-length+cDNA+clones.">Large-scale RT-PCR recovery of full-length cDNA clones.</a> Biotechniques. 36, 690-696 (2004).</li><li>Wu, J. Q., Shteynberg, D., Arumugam, M., Gibbs, R. A., Brent, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+rat+genes+by+TWINSCAN+gene+prediction,+RT-PCR,+and+direct+sequencing.">Identification of rat genes by TWINSCAN gene prediction, RT-PCR, and direct sequencing.</a> Genome Res. 14, 665-671 (2004).</li><li>Dewey, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+identification+of+novel+human+genes+through+simultaneous+gene+prediction+in+human,+mouse,+and+rat.">Accurate identification of novel human genes through simultaneous gene prediction in human, mouse, and rat.</a> Genome Res. 14, 661-664 (2004).</li><li>Wu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Characterize Mammalian Transcriptome Complexity. , (2011).</li></ol>

The authors have nothing to disclose.

Mammalian transcriptome is very complex34-38. RNA-Seq technology plays an increasingly important role in the studies of transcriptome analysis, novel transcripts detection and single nucleotide variation discovery etc. It has many advantages over other methods for gene expression analysis. As mentioned in the introduction, it overcomes the hybridization artifacts of microarray and can be used to identify novel transcripts de novo. One limitation of RNA-sequencing is relative short read length comparing to Sanger sequencing. However, with the rapid improvement of sequencing technology, read length is increasing constantly. In this paper, we provide detailed methods of using this technology to identify potential key regulators in mouse EML cell self-renewal and differentiation.
The first key step for this protocol is EML cell culture. Although EML is a hematopoietic precursor cell line and it can be propagated in large quantity with SCF. The culturing condition of EML cells requires more attention than the usual immortalized cell lines. The cells should be fed and passaged at a regular basis with gentle operation; otherwise the cells could change in their properties of self-renewal and differentiation and undergo cell death. As the first step after collecting enough cells, we isolated lineage negative cells using a magnetic activated cell sorting system. Then we separated CD34+ and CD34- cells using fluorescence-activated cell sorting. The EML cells are normally passaged less than 10 generations before using for RNA extraction and the numbers of CD34+ and CD34- cells should be similar after separation. If the two populations vary greatly in cell number, it is advisable to discard the culture and re-thaw another tube of cell stock for culture.
After separation of CD34+ and CD34- cell, total RNA extraction was performed, another important step for this study. High quality RNA is the base for construction of a high quality library, which promises the accuracy of the sequencing data. In this critical step, any contact with RNase should be avoided. All reagents should be RNase free. It is important to wear gloves at all times while handling RNA. High quality RNA sample has an OD 260/280 value between 1.8 and 2.0. When collecting the aqueous phase containing RNA, be careful not to carry any organic phase with the RNA sample. Any residual organic solvents such as phenol or chloroform in the RNA would result in an OD260/280 value lower than 1.65. If the OD260/280 value is lower than 1.65, precipitate RNA again with ethanol. After washing with 75% ethanol, do not overdry RNA pellet. Drying RNA pellet completely will affect the solubility of RNA and lead to low yield of RNA.
The next key step for this protocol is library preparation. After total RNA extraction, a step of using DNase for removal of contaminated DNA is highly recommended, since DNA contamination might result in wrong estimation of the amount of total RNA used. It is recommended to perform the downstream procedure immediately after RNA isolation, since after long-term storage and freeze-thawing procedure, RNA will degrade to some degree. If the subsequent steps after RNA isolation can not be performed immediately, store the RNA in -80 &#176;C. Before total RNA is used for mRNA purification and cDNA synthesis, the quality should always be checked. Only high quality RNA can be used for library preparation. Using low quality or degraded RNA might lead to over-representation of 3&#39; ends. Before sequencing, library quality was assessed to ensure maximum sequencing efficiency.
In the data analysis part, after performing a run of Cufflinks without a reference transcriptome, we combined the novel transcripts with known transcripts to form a reference .gtf file and run Tophat and cufflinks for the second time. This two-run procedure is recommended, since this provide more accurate FPKM estimation than running only once. After data analysis, the differentially expressed genes were identified. Downstream experiments can be performed to validate the function of genes in vitro and in vivo. In our previous publication2, we chose the significantly differentially expressed transcription factors and identified the genome binding site of these factors by performing chromatin immunoprecipitation and sequencing (ChIP-Seq). In addition, we applied shRNA knockdown assay to test the functional effect of Tcf7. We found that in Tcf7 knockdown cells, up-regulated genes were the genes highly enriched in CD34- cells, while down-regulated genes were found to be significantly enriched in CD34+ cells. Therefore, the gene expression profile of Tcf7 knockdown cells shifted toward a partially differentiated CD34- state.Overall, using EML cell as a model system coupled with RNA-Sequencing technology and functional assays, we identified and confirmed Tcf7 as an important regulator of EML cell self-renewal and differentiation.

Hematopoietic stem cells are rare blood cells that reside mainly in the adult bone marrow niche. They are responsible for the production of cells required to replenish the blood and the immune systems1. As a kind of stem cells, HSCs are capable of both self-renewal and differentiation. Elucidating mechanisms that control the fate decision of HSCs, toward either self-renewal or differentiation, will offer valuable guidance on the manipulation of HSCs for blood disease researches and clinical usage2. One problem faced by the researchers is that HSCs can be maintained and expanded in vitro to a very limited extent; the vast majority of their progeny are partially differentiated in culture2.
In order to identify key regulators that control the processes of self-renewal and differentiation at a genome-wide scale, we used a mouse primitive hematopoietic progenitor cell line EML as a model system. This cell line was derived from murine bone marrow3,4. When fed with different growth factors, EML cells can differentiate into erythroid, myeloid, and lymphoid cells in vitro5. Importantly, this cell line can be propagated in large quantity in culture medium containing stem cell factor (SCF) and still retaining their multipotentiality. EML cells can be separated into subpopulations of self-renewing Lin-SCA+CD34+ and partially differentiated Lin-SCA-CD34- cells based on surface markers CD34 and SCA6. Similar to short-term HSCs, SCA+CD34+ cells are able of self-renewal. When treated with SCF, Lin-SCA+CD34+ cells can rapidly regenerate a mixed population of Lin-SCA+CD34+ and Lin-SCA-CD34- cells and continue to proliferate6. The two populations are similar in morphology and have similar levels of c-kit mRNA and protein6. Lin-SCA-CD34- cells are capable of propagating in media containing IL-3 instead of SCF3. Unveiling the key regulators in the EML cell fate decision will offer better understanding of cellular and molecular mechanisms in early developmental transition during hematopoiesis.
In order to investigate the underlying molecular differences between the self-renewing Lin-SCA+CD34+ and partially differentiated Lin-SCA-CD34- cells, we used RNA-Seq to identify differentially expressed genes. In particular, we focus on transcription factors, as transcription factors are crucial in determining cell fate. RNA-Seq is a recently developed approach that utilizes the capabilities of next-generation sequencing (NGS) technologies to profile and quantify RNAs transcribed from genome7,8. In brief, total RNA is poly-A selected and fragmented as the initial template.The RNA template is then converted into cDNA using reverse transcriptase. In order to map full-length RNA transcripts, using intact, non-degraded RNA for constructing cDNA library is important. For the purpose of sequencing, specific adapter sequences are added to both ends of cDNA. Then, in most cases, cDNA molecules are amplified by PCR and sequenced in a high-throughput manner.
After sequencing, the resulting reads can be aligned to a reference genome and a transcriptome database. The number of reads that map to the reference gene is counted and this information can be used to estimate the gene expression level. The reads can also be assembled de novo without a reference genome, enabling the study of transcriptomes in non-model organisms9. RNA-seq technology has also been used to detect splice isoforms10-12, novel transcripts13 and gene fusions14. In addition to the detection of protein-coding genes, RNA-Seq can also be used to detect novel and analyze transcription level of non-coding RNAs, such as long non-coding RNA15,16, microRNA17, siRNA etc.18. Because of the accuracy of this method, it has been utilized for detection of single nucleotide variations19,20.
Before the advent of RNA-Seq technology, microarray was the main method used for analyzing gene expression profile. Pre-designed probes are synthesized and subsequently attached to a solid surface to form a microarray slide21. mRNA is extracted and converted to cDNA. During the reverse transcription process, fluorescently labeled nucleotides are incorporated into the cDNA and the cDNA can be hybridized onto the microarray slides. The intensity of the signal collected from a specific spot depends on the amount of cDNA binding to the specific probe on that spot21. Compared with RNA-Seq technology, microarray has several limitations. First, microarray relies on the pre-existing knowledge of gene annotation, while RNA-Seq technology is able to detect novel transcripts at relative high background level, which limits its use when gene expression level is low. Besides, the RNA-Seq technology has much higher dynamic range of detection (8,000 fold)7, whereas, due to background and saturation of signals, the accuracy of microarray is limited for both highly and lowly expressed genes7,22. Finally, microarray probes differ in their hybridization efficiencies, which make the results less reliable when comparing relative expression levels of different transcripts within one sample23. Although RNA-Seq has many advantages over microarray, its data analysis is complex. This is one of the reasons that many researchers still use microarray instead of RNA-Seq. Various bioinformatics tools are required for RNA-Seq data processing and analysis24.
Among several next-generation sequencing (NGS) platforms, 454, Illumina, SOLID and Ion Torrent are the most widely used ones. 454 was the first commercial NGS platform. In contrast to the other sequencing platforms such as illumina and SOLID, the 454 platform generates longer read length (average 700 base reads)25. Longer reads are better for initial characterization of transcriptiome due to their higher assemble efficiency25. The main disadvantage of the 454 platform is its high cost per megabase of sequence. The Illumina and SOLID platforms generate reads with increased numbers and short lengths. The cost per megabase of sequence is much lower than the 454 platform. Due to the large numbers of short reads for the Illumina and SOLID platforms, data analysis is much more computationally intensive. The price of the instrument and reagents for sequencing for the Ion Torrent platform is cheaper and the sequencing time is shorter25. However, the error rate and the cost per megabase of sequence are higher compared to the Illumina and SOLID platforms. Different platforms have their own advantages and disadvantages and require different methods for data analysis. The platform should be chosen based on the sequencing purpose and the availability of funding.
In this paper, we take Illumina RNA-Seq platform as an example. We used EML cell as a model system to investigate the key regulators in EML cell self-renewal and differentiation, and provided a detailed methods of RNA-Seq library construction and data analysis for expression level calculation and novel transcript detection. We have shown in our previous publication that RNA-seq study in EML model system2, when coupled with functional test (e.g. shRNA knockdown) provide a powerful approach in understanding the molecular mechanism of the early stages of hematopoietic differentiation, and can serve as a model for the analysis of cell self-renewal and differentiation in general.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>Invitrogen</td>
			<td>15240-062</td>
			<td>BHK cell culture</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD34 FITC</td>
			<td>eBioscience</td>
			<td>11-0341-81</td>
			<td>FACS sorting</td>
		</tr>
		<tr>
			<td>Anti-Mouse Ly-6A/E (Sca-1) PE</td>
			<td>eBioscience</td>
			<td>12-5981-81</td>
			<td>FACS sorting</td>
		</tr>
		<tr>
			<td>APC Mouse Lineage Antibody Cocktail</td>
			<td>BD Biosciences</td>
			<td>558074</td>
			<td>FACS sorting</td>
		</tr>
		<tr>
			<td>BD FACSAria Cell Sorter</td>
			<td>BD Biosciences</td>
			<td>Special offer sysmtem</td>
			<td>FACS sorting</td>
		</tr>
		<tr>
			<td>Corning&#8482; Cell Culture Treated Flasks 75cm2</td>
			<td>Corning incorporated</td>
			<td>430641</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Corning&#8482; Cell Culture Treated Flasks 25cm2</td>
			<td>Corning incorporated</td>
			<td>430639</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I, Amplification Grade</td>
			<td>Invitrogen</td>
			<td>18068-015</td>
			<td>Library preparation</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>BHK cell culture</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>HI FBS&#160;</td>
			<td>Invitrogen</td>
			<td>16140071</td>
			<td>BHK cell culture</td>
		</tr>
		<tr>
			<td>Horse Serum</td>
			<td>Invitrogen</td>
			<td>16050-122</td>
			<td>EML cell culture</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>HyClone</td>
			<td>SH30228.02</td>
			<td>EML cell culture</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Invitrogen</td>
			<td>25030-081</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Lineage Cell Depletion Kit, mouse&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-090-858</td>
			<td>Isolation of lineage negative cells</td>
		</tr>
		<tr>
			<td>NanoVue Plus spectrophotometer&#160;</td>
			<td>GE Healthcare</td>
			<td>28-9569-62</td>
			<td>Quality control</td>
		</tr>
		<tr>
			<td>Thermo Scientific&#8482; Napco&#8482; 8000 Water-Jacketed CO2 Incubators</td>
			<td>Thermo Scientific</td>
			<td>15-497-002&#160;</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140-122</td>
			<td>EML cell culture</td>
		</tr>
		<tr>
			<td>TRIzol&#174; Reagent</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
			<td>RNA exraction</td>
		</tr>
		<tr>
			<td>TruSeq&#8482; RNA Sample Prep Kit v2 -Set B (48rxn)</td>
			<td>Illumina</td>
			<td>RS-122-2002</td>
			<td>Library preparation</td>
		</tr>
		<tr>
			<td>2100 Electrophoresis Bioanalyzer Instrument&#160;</td>
			<td>Agilent</td>
			<td>G2939AA</td>
			<td>Quality control</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>0.45 &#181;m Syringe Filters</td>
			<td>Nalgene</td>
			<td>190-2545</td>
			<td>Cell culture</td>
		</tr>
	</tbody>
</table>

identification of key factors regulating self-renewal and differentiation in eml hematopoietic precursor cells by rna-sequencing analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

In order to analyze differentially expressed genes in Lin-CD34+ and Lin-CD34- EML cells, we used RNA-Seq technology. Figure 1 shows the workflow of the procedures. After isolation of lineage negative cells by magnetic cell sorting, we separated Lin-SCA+CD34+ and Lin-SCA-CD34- cells using FACS Aria. Lin-enriched EML cells were stained with anti-CD34, anti-Sca1 and lineage cocktail antibodies. Only Lin- cells were gated for analysis of Sca1 and CD34 expression. Two populations (SCA+CD34+ and SCA-CD34- EML cells) could be observed by FACS analysis (Figure 2)6.
After cell separation, we extracted total RNA from CD34+ and CD34- cells respectively and analyzed the quality of RNA. The accuracy of RNA-Seq data largely relies on the quality of RNA-Seq library and the quality of total RNA is vital for preparing a high quality library. High quality RNA sample should have an OD 260/280 value between 1.8 and 2.0. In addition to using the spectrophotometer, RNA quality was further assessed with greater accuracy by Bioanalyzer. Figure 3 shows a result of a high quality RNA sample with the RIN equal to 9.4. Only high quality total RNA sample with RIN value greater than 9 was used for mRNA extraction and subsequent library construction procedures.
Ribosomal RNA is the most abundant type of RNA in cell. Currently two main strategies, depletion of rRNA or positively selection of polyadenylated mRNA (poly-A mRNA), are used for enrichment of target RNA before library construction. Non polyadenylated RNA species are lost during the selection of poly-A mRNA. In contrast, rRNA depletion methods such as RiboMinus could preserve non polyadenylated RNA species. The purpose of our study is to look for differentially expressed coding genes in two cell types, thus we used the poly-A mRNA selection method for enrichment of target RNAs before library construction. When library construction was finished, the size of DNA fragments in the library was checked before sequencing using Bioanalyzer. Figure 4 shows a good quality library with the fragment size peaks at about 300 bp.
In the subsequent step, the library was subjected to high-throughput sequencing. In principle, longer read length will be helpful for read mapping. It can reduce the probability that the read is mapped to multiple locations due to similarity among duplicate genes or gene family members. As the pair-end sequencing sequences are from both ends of the fragments, the read length chosen should be less than half of the average fragments length. If the main goal of the experiment is to measure the expression level instead of constructing transcript structure, single-end read (75 or 100 bp) can reduce the cost without losing too much information. Paired-end sequencing is more useful for transcript structure construction and shorter read length can be used to reduce cost. Certainly, when sufficient funding is available, longer read length is preferred.
For differential expression analysis, there are many alternative algorithms other than DESeq. There is also one included in cufflinks package named cuffdiff32. DESeq is one of the most widely used count based DE gene analysis algorithms. DESeq method is based on a well characterized statistics model &#8212; negative binomial distribution. In our experience, DESeq is more stable compare to cuffdiff. Early versions of cuffdiff often give significantly different numbers of DE genes. Therefore we used DESeq for DE analysis here.
Because transcription factors are crucial for cell fate determination, we focused on the significantly differentially expressed transcription factors33. The TFs changed &#62;1.5 fold between Lin-CD34+ and Lin-CD34- were found and are shown on the heatmap (Figure 5)2. Notably, the relative expression level of Tcf7 in Lin-CD34+ cells is more than 100 fold higher than that in Lin-CD34- cells. Thus Tcf7 was chosen for further ChIP-Sequencing (Chromatin Immunoprecipitation and sequencing) analysis and functional test to confirm Tcf7&#8217;s function in regulation of EML cell self-renewal and differentiation2.
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/52104/52104fig1highres.jpg" /> 
Figure 1: Workflow of the procedures. Lin-CD34+ and Lin-CD34- cells were separated by magnetic cell separation system and fluorescence-activated cell sorting method. Total RNA was extracted followed by mRNA purification and library construction. After analysis of library quality, samples were subjected to high throughput sequencing. Data were analyzed and differentially expressed transcription factors were identified.
<img alt="Figure 2" fo:content-width="2.7in" src="/files/ftp_upload/52104/52104fig2highres.jpg" width="300" /> 
Figure 2: Separation of Lin-CD34+ and Lin-CD34- EML cells6. Lin- EML cells were enriched by magnetic cell sorting. Lin- cells were stained with anti-CD34, anti-Sca1 and lineage mixture antibodies. Lin- cells were gated for expression of CD34 and Sca1. Lin-CD34+SCA+ and Lin-CD34-SCA- EML cell populations were sorted.
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/52104/52104fig3highres.jpg" width="600" /> 
Figure 3: A representative of high-quality total RNA sample. The quality of total RNA was assessed by Bioanalyzer. The RNA Integrity Number is 9.4 (FU, Fluorescence Units).
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/52104/52104fig4highres.jpg" width="600" /> 
Figure 4: Fragments size range of Paired-End library. The DNA size distribution of the library was analyzed using Bioanalyzer. Most fragments are within the size range of 250-500 bp.
<img alt="Figure 5" fo:content-width="2.8in" src="/files/ftp_upload/52104/52104fig5highres.jpg" width="300" /> 
Figure 5: Differentially expressed transcription factors (&#62;1.5 fold) between Lin-CD34+ cells and Lin-CD34- cells2. For each cell type, two independent experiments were performed. Up-regulated genes are indicated as red color and down-regulated genes are indicated as green color.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="2">BHK medium</td>
		</tr>
		<tr>
			<td>100x Antibiotic-Antimycotic&#160;</td>
			<td>10 ml&#160;&#160;</td>
		</tr>
		<tr>
			<td>200 mM L-Glutamine&#160;</td>
			<td>10 ml&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>100 ml&#160;&#160;</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>880 ml</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>1,000 ml</td>
		</tr>
		<tr>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td colspan="2">EML Basic medium</td>
		</tr>
		<tr>
			<td>IMDM</td>
			<td>390 ml</td>
		</tr>
		<tr>
			<td>HI horse serum</td>
			<td>100 ml</td>
		</tr>
		<tr>
			<td>100 x Penicillin-Streptomycin</td>
			<td>5 ml</td>
		</tr>
		<tr>
			<td>200 mM L-Glutamine</td>
			<td>5 ml</td>
		</tr>
		<tr>
			<td>BHK medium</td>
			<td>75 ml</td>
		</tr>
		<tr>
			<td>Total Volume</td>
			<td>575 ml</td>
		</tr>
		<tr>
			<td colspan="2">Filtrate through 0.45 &#956;M filter</td>
		</tr>
		<tr>
			<td colspan="2"></td>
		</tr>
		<tr>
			<td colspan="2">FACS buffer</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>0.50%</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td colspan="2">Dissolved in PBS and filtrate through 0.45 &#956;M filter</td>
		</tr>
	</tbody>
</table>
Table 1: Buffers and Cell culture mediums.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Software&#160;</td>
			<td>Usage</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>Bowtie 1.2.7</td>
			<td>Used by Tophat for mapping</td>
			<td>[28]</td>
		</tr>
		<tr>
			<td>Tophat 1.3.3</td>
			<td>Mapping reads back to reference genome</td>
			<td>[27]</td>
		</tr>
		<tr>
			<td>Cufflinks 1.3.0</td>
			<td>Transcripts construction and expression level estimation</td>
			<td>[29]</td>
		</tr>
		<tr>
			<td>DESeq 1.16.0</td>
			<td>Differential expression analysis</td>
			<td>[30]</td>
		</tr>
		<tr>
			<td>Bedtools 2.18</td>
			<td>Convert .bam file into .bed file</td>
			<td>[31]</td>
		</tr>
		<tr>
			<td>bedGraphToBigWig</td>
			<td>Convert .bed file to .bigwig file</td>
			<td><a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a></td>
		</tr>
	</tbody>
</table>
Table 2: List of software for data analysis.

Watch this Scientific Journal Video about Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis at JoVE.com

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

identification-key-factors-regulating-self-renewal-differentiation

Nanyang Technological University

Research

JoVE Journal

Biology

12.3K Views.  The University of Texas Graduate School of Biomedical Sciences at Houston. RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Video: Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

The use of SC1 (Pluripotin) to Support mESC Self-renewal in the Absence of LIF

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and activation of the LIF/JAK/STAT3 pathway is not absolutely required to maintain the self-renewal state.2 The SC1 small molecule may be an economical alternative to LIF. SC1 functions through dual inhibition of Ras-GAP and ERK1.3 Illustration of its mechanism of action makes it a useful tool to study the fundamental molecular mechanism of self-renewal. Here we demonstrate the procedure for culturing mouse ES cells in the presence of SC1 and show that they are able to maintain self-renewal in the absence of LIF. Cells cultured with SC1 showed similar morphology compared to cells maintained with LIF. Both exhibited typical mouse ES morphology after five passages. Expression of typical pluripotency markers (Oct4, Sox2, Nanog, and SSEA1) was observed after five passages in the presence of SC1. Furthermore, SC1 caused no overt toxicity on mouse ES cells.

The use of SC1 Pluripotin to Support mESC Self-renewal in the Absence of LIF

SC1 functions through dual inhibition of Ras- GAP and ERK1. We tested the function of SC1 in supporting mouse ES cell self-renewal in the absence of LIF and showed that SC1 is able to maintain self-renewal of mouse ES cell cultures.

Mouse embryonic stem (ES) cells are conventionally cultured with Leukemia Inhibitory Factor (LIF) to maintain self-renewal.1 However, LIF is expensive and ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, small ubiquitin-related modifier (SUMO) is a ubiquitin-like protein that is covalently attached to the lysine residues of a variety of target proteins to regulate cellular processes, such as gene transcription, DNA repair, protein interaction and degradation, subcellular transport, and signal transduction. The most common approach to detecting protein SUMOylation is based on the expression and purification of recombinant tagged proteins in bacteria, allowing for an in vitro biochemical reaction which is simple and suitable for addressing mechanistic questions. However, due to the complexity of the process of SUMOylation in vivo, it is more challenging to detect and analyze protein SUMOylation in cells, especially when under endogenous conditions. A recent study by the authors of this paper revealed that endogenous retinoblastoma (Rb) protein, a tumor suppressor that is vital to the negative regulation of the cell cycle progression, is specifically SUMOylated at the early G1 phase. This paper describes a protocol for the detection and analysis of Rb SUMOylation under both endogenous and exogenous conditions in human cells. This protocol is appropriate for the phenotypical and functional investigation of the SUMO-modification of Rb, as well as many other SUMO-targeted proteins, in human cells.

In Vivo Detection and Analysis of Rb Protein SUMOylation in Human Cells

Small ubiquitin-related modifier (SUMO) family proteins are conjugated to the lysine residues of target proteins to regulate various cellular processes. This paper describes a protocol for the detection of retinoblastoma (Rb) protein SUMOylation under endogenous and exogenous conditions in human cells.

The post-translational modifications of proteins are critical for the proper regulation of intracellular signal transduction. Among these modifications, ...