Funding for this work was provided by the University of San Diego, the National Science Foundation Graduate Research Fellowship Program, NIH grant R01-HG007836, and by the Korean Ministry of Science, ICT and Future Planning.
Earlier versions of a several figures were first published in "Hoople, G. D. et al. Gel-seq: whole-genome and transcriptome sequencing by simultaneous low-input DNA and RNA library preparation using semi-permeable hydrogel barriers. Lab on a Chip 17, 2619-2630, doi:10.1039/c7lc00430c (2017)." Lab on a Chip has sanctioned the reuse of figures in this publication.

<ol>
	<li>Mawy, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+sequencing.">Single-cell sequencing.</a> Nat Methods. 11 (1), 18 (2014).</li><li>Gole, 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=Massively+parallel+polymerase+cloning+and+genome+sequencing+of+single+cells+using+nanoliter+microwells.">Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells.</a> Nat Biotech. 31 (12), 1126-1132 (2013).</li><li>Sasagawa, Y., 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=Quartz-Seq:+a+highly+reproducible+and+sensitive+single-cell+RNA-Seq+reveals+non-genetic+gene+expression+heterogeneity.">Quartz-Seq: a highly reproducible and sensitive single-cell RNA-Seq reveals non-genetic gene expression heterogeneity.</a> Genome Biol. 14 (4), 31 (2013).</li><li>Ramsk&#246;ld, 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=Full-length+mRNA-Seq+from+single-cell+levels+of+RNA+and+individual+circulating+tumor+cells.">Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells.</a> Nat Biotechnol. 30 (8), 777-782 (2012).</li><li>Shapiro, E., Biezuner, T., Linnarsson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+sequencing-based+technologies+will+revolutionize+whole-organism+science.">Single-cell sequencing-based technologies will revolutionize whole-organism science.</a> Nat Rev Genet. 14 (9), 618-630 (2013).</li><li>Navin, N., 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=Tumour+evolution+inferred+by+single-cell+sequencing.">Tumour evolution inferred by single-cell sequencing.</a> Nature. 472 (7341), 90-94 (2011).</li><li>Hoople, G. 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=Gel-seq:+whole-genome+and+transcriptome+sequencing+by+simultaneous+low-input+DNA+and+RNA+library+preparation+using+semi-permeable+hydrogel+barriers.">Gel-seq: whole-genome and transcriptome sequencing by simultaneous low-input DNA and RNA library preparation using semi-permeable hydrogel barriers.</a> Lab Chip. 17, 2619-2630 (2017).</li><li>Macaulay, I. 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=G&amp;T-seq:+parallel+sequencing+of+single-cell+genomes+and+transcriptomes.">G&amp;T-seq: parallel sequencing of single-cell genomes and transcriptomes.</a> Nat Methods. 12 (6), 519-522 (2015).</li><li>Dey, S. S., Kester, L., Spanjaard, B., Bienko, M., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrated+genome+and+transcriptome+sequencing+of+the+same+cell.">Integrated genome and transcriptome sequencing of the same cell.</a> Nat Biotechnol. 33 (3), 285-289 (2015).</li><li>Gopal, A., Zhou, Z. H., Knobler, C. M., Gelbart, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+large+RNA+molecules+in+solution.">Visualizing large RNA molecules in solution.</a> RNA. 18 (2), 284-299 (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SDS-PAGE+Gel.">SDS-PAGE Gel.</a> Cold Spring Harb Protoc. 2015 (7), 087908 (2015).</li><li>Lake, B. B., 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=Neuronal+subtypes+and+diversity+revealed+by+single-nucleus+RNA+sequencing+of+the+human+brain.">Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain.</a> Science. 352 (6293), (2016).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. -. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+Gel+Electrophoresis+for+the+Separation+of+DNA+Fragments.">Agarose Gel Electrophoresis for the Separation of DNA Fragments.</a> J Vis Exp. (62), e3923 (2012).</li><li>Qubit 3.0 Fluorometer Manual. MAN0010866. Life Technologies Available from: <a target="_blank" href="https://tools.thermofisher.com/content/sfs/manuals/qubit_3_fluorometer_man.pd">https://tools.thermofisher.com/content/sfs/manuals/qubit_3_fluorometer_man.pd</a> (2017)</li><li>Vitak, S. 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=Sequencing+thousands+of+single-cell+genomes+with+combinatorial+indexing.">Sequencing thousands of single-cell genomes with combinatorial indexing.</a> Nat Methods. 14 (3), 302-308 (2017).</li><li>Illumina. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nextera &#174; XT DNA Library Preparation Kit. , (2017).</li><li>Takara Bio USA Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing User Manual. , (2016).</li><li>Kurimoto, K., Yabuta, Y., Ohinata, Y., Saitou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+single-cell+cDNA+amplification+to+provide+a+template+for+representative+high-density+oligonucleotide+microarray+analysis.">Global single-cell cDNA amplification to provide a template for representative high-density oligonucleotide microarray analysis.</a> Nat Protoc. 2 (3), 739-752 (2007).</li></ol>

KZ is co-founder and Scientific Advisor of Singlera Genomics Inc.

There are several critical steps associated with the Gel-seq device fabrication as well as the protocol itself. During fabrication, we recommend starting with the prescribed layer thicknesses for the various regions of the gel. We spent significant time testing different fabrication options and the protocol described here produces the best devices for the cassettes listed in the Table of Materials and Reagents. If researchers use an alternative cassette system, they may find it necessary to tweak the volumes used when creating the devices. The major challenge in fabrication is that if the high-density gel region is too large it can delaminate from the edges of the cassette and create air pockets on the interior of the cassette that will disrupt electrophoresis. By casting several cassettes with several different layer volumes, researchers should be able to quickly determine the optimal configuration for their specific hardware.
The Gel-seq protocol also has several critical steps that can be validated before the protocol is complete. One potential failure point is the separation of gDNA and RNA/cDNA hybrids. This can be validated by imaging the Gel-seq device after separation (see Figure 3B). In one set of experiments, we found that our lab supply of buffer had become contaminated with DNA and was causing substantial autoflourescence in our device (see Figure 3D). This made it difficult to determine if the separation had taken place. Fluorescent imaging helped us identify and correct this problem before using any costly reagents to generate sequencing libraries.
Another critical point is step 6.2, the qPCR amplification of cDNA after separation. Researchers should pay careful attention not to overamplify in this step as it will reduce the quality of the RNA-seq data. This consideration is not unique to Gel-seq, but is a common aspect of low-input RNA-seq library preparation. PCR amplification during sequencing library preparation is often necessary, but it can introduce sequence errors and biases. The required number of cycles for PCR depends on sample quantity and complexity. It is generally advisable to limit PCR cycle number to the bare minimum required to yield sufficient clustering when the libraries are sequenced. In theory, a protocol can be optimized to determine the exact cycle number that yields sufficient copy number without introducing excessive artifacts. In practice, however, inconsistencies in sample quality, loading, or handling early in the protocol can dramatically affect the distribution of molecular templates available for library prep PCR, which in turn affects the optimal PCR cycle number. The most general solution we have found to monitor the progress of the amplification reactions using a fluorescent dye, run the reactions on a real-time PCR thermocycler, and stop the reactions in the exponential (linear versus cycle number) phase. In our experience, real-time monitoring is especially relevant when developing, adapting, or adopting a new protocol.
The last critical step is generating genomic and transcriptomic libraries. The key to this step is to set the starting sample concentration for the DNA library prep reaction as close to 0.2 ng/&#181;L (0.5 ng total) as possible. This is relatively straightforward for the qPCR amplified cDNA as there is usually an excess of cDNA, but it can be more challenging for the gDNA samples. We found careful attention to the vacufuge step was required while the samples were being concentrated. As expected, in experiments with 1000 cells, the vacufuge step could be stopped much sooner than experiments with 100 cells. The number of samples in the vacufuge also impacted the evaporation rate in our experiments. We found that using a flourometer to validate DNA content midway through the concentration step could be helpful when performing the protocol with unfamiliar samples. Fortunately, if researchers over concentrate a sample, nuclease free water can be added to dilute the sample. Theoretically, it is possible to use the vacufuge to dry the DNA and then resuspend it in the desired volume; however, we suggest avoiding complete evaporation.
We view the three current methods for generating simultaneous DNA and RNA libraries, Gel-seq7, G&#38;T-seq8, and DR-seq9, as complimentary. Gel-seq is ideal for samples in the 100 - 1000 cell range and requires no pull-down targets or a priori knowledge of the genome. The other two methods are better suited for single cell applications. One of our goals in developing Gel-seq was to create a protocol that could be easily implemented by other researchers. We therefore decided to fabricate devices within the standard form factor of a polyacrylamide gel cassette. While the technique we used to define our different membranes is novel, most genetics labs already have all the necessary equipment to fabricate the Gel-seq device. Furthermore, the cost of the device is trivial - just $5.25 for a device that can process 12 samples. That said, as with any library preparation protocol using commercial reagents, the overall cost for generating libraries remains high. Our reagent cost per sample was $50 for whole-transcript amplification and $28 for library preparation for both DNA and RNA. Fortunately, the Gel-seq device itself is protocol agnostic. For example, during development we successfully tested the device using cells from culture and an older RNA library amplification protocol19, although we found it was not suitable for tissue samples from mice. Looking towards the future, as cheaper alternatives for library preparation are developed, our protocol can be adapted to work with these new techniques. We believe researchers will find it straightforward to implement Gel-seq in their own labs. We hope this will facilitate the rapid adoption of the technology.

Next generation sequencing (NGS) has had a profound impact on the way genetics research is conducted. Where researchers once focused on sequencing the genome of an entire species, it is now possible to sequence the genome of a single tumor or even a single cell in one experiment.1 NGS has also made it cost effective to sequence the RNA transcripts found within a cell, a collection of data known as the transcriptome. The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years.2,3,4 Unfortunately, standard protocols are incompatible and researchers must choose whether to sequence DNA or RNA for a given sample. When a starting sample is large enough, it can be split in half. At smaller scales, however, loss of material due to splitting samples can affect library quality, and pooling of samples can average out interesting variations between cells.5 Furthermore, researchers are increasingly interested in examining samples that cannot be split, such as single cells or small heterogeneous tumor biopsies.6
To address this problem, three protocols have recently been developed to sequence both DNA and RNA from the same starting sample: Gel-seq7, G&#38;T-seq8, and DR-seq9. This article presents a detailed protocol for Gel-seq, which can be used to simultaneously generate DNA and RNA libraries from as few as 100 cells at negligible added cost. The novel aspect of Gel-seq is the ability to separate DNA and RNA based exclusively on size using low cost hydrogel matrices. The core innovation of the Gel-Seq protocol is the physical separation of DNA from RNA. This separation is achieved electrophoretically using a combination of polyacrylamide membranes that take advantage of the size differences between these molecules. To put these size differences in context, consider how DNA and RNA are imaged: while DNA exists on the micron-scale and can be viewed using traditional microscopes, RNA exists on the nanometer scale and must be imaged using complex techniques such as cryo-electron microscopy.10
The approach to separating DNA and RNA in this protocol is shown in Figure 1. The left panel shows DNA and RNA free floating in solution near a membrane. When an electric field is applied, as shown in the right panel, DNA and RNA experience an electrophoretic force that induces migration through the membrane. By tuning the membrane properties, we have created a semi-permeable membrane that separates DNA from RNA. The DNA molecules are pushed against the membrane, but become entangled at the edge because of their large size. Small RNA molecules, on the other hand, can reconfigure and weave their way through the membrane. This process, known as reptation, is similar to the way a snake moves through grass. Eventually these RNA molecules are stopped by a second, high-density membrane that is too difficult for even smaller polymers (&#62;200 base pairs) to wriggle through. Once physically separated, DNA and RNA can be recovered and processed to generate information about both the genome and transcriptome. While we can separate DNA and RNA, we have found better results are obtained if the RNA is reverse transcribed to cDNA before separation. The cDNA/RNA hybrids are more stable than RNA alone and can still pass through the low-density membrane.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57315/57315fig1.jpg" /> 
Figure 1. Gel-seq Operating Principle.&#160;The underlying principle used to physically separate DNA and RNA. In an applied electric field, small RNA molecules migrate through the low-density membrane but large DNA molecules are trapped at the surface. This figure was reproduced from Ref. 7 with permission from the Royal Society of Chemistry.&#160;<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57315/57315fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This paper describes in detail both the fabrication of the Gel-seq device and the biological protocol to generate paired DNA and RNA libraries. An overview of both is shown in Figure 2. The device is fabricated by layering three different density polyacrylamide gels on top of each other in a process similar to creating standard stacking gels.11 The biological protocol starts with 100 - 1000 cells suspended in PBS. The cells are lysed and the RNA is converted into cDNA before the device is used to separate the genomic DNA from the cDNA/RNA hybrids. After separation and recovery, genomic and transcriptomic libraries are prepared using a process that closely follows the standard whole-genome library preparation kit protocol. Further detail about the development and validation of Gel-seq can be read in the Lab on a Chip publication &#34;Gel-seq: whole-genome and transcriptome sequencing by simultaneous low-input DNA and RNA library preparation using semi-permeable hydrogel barriers.&#34;7
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57315/57315fig2.jpg" /> 
Figure 2. Gel-seq Protocol.&#160;An overview of the steps to fabricate the Gel-seq device and the protocol to generated paired DNA and RNA libraries. Portions of this figure were reproduced from Ref. 7 with permission from the Royal Society of Chemistry.&#160;<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57315/57315fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To generate DNA and RNA libraries from single cells, researchers should consider using either G&#38;T-seq or DR-seq. G&#38;T-Seq, like Gel-seq, relies on a physical separation of RNA from genomic DNA. This approach relies on messenger RNA&#39;s (mRNA) 3&#8242; polyadenylated tail as a pull-down target. The mRNA is captured on a magnetic bead using a biotinylated oligo-dT primer. Once the mRNA has been captured the beads are held in place with a magnet and the supernatant containing the genomic DNA can be removed and transferred to another tube. After this physical separation is complete, separate libraries can be generated from the mRNA and DNA.8 This approach works well if the RNA of interest is polyadenylated, however it cannot be used to study non-polyadenylated transcripts, such as ribosomal RNA, tRNA, or RNA from prokaryotes.
DR-seq relies on a pre-amplification step where both DNA and cDNA derived from RNA are amplified in the same tube. The sample is then split in two and processed in parallel to prepare DNA- and RNA-seq libraries. To distinguish between genomic DNA and the cDNA derived from RNA, DR-seq takes a computational approach. Sequences where only exons are present are computationally suppressed in the genomic DNA data, as those could have originated from either DNA or RNA.9 An advantage of this approach is that the DNA and cDNA/RNA need not be physically separated as is done in Gel-seq and G&#38;T-seq. The drawback, however, is that DR-seq requires a priori knowledge of the genome and transcriptome (i.e., exons versus introns), and might not be ideal for applications such as sequencing of nuclei, in which many transcripts are not yet fully spliced and still contain introns.12
The novel aspect of Gel-seq is the ability to separate DNA and RNA in hundreds of cells based exclusively on size. This method requires no a priori knowledge of the genome or transcriptome, is robust against incomplete splicing, and is not limited to poly-adenylated transcripts. For applications where a researcher can start with at least 100 cells, Gel-seq provides a straightforward approach using cheap and widely-available materials.

<table border="0" cellpadding="0" cellspacing="0" style="width:905px;" width="906">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Reagents</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Acrylamide Monomer</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">A8887-100G</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Ammonium Persulfate</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">A3678-25G</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Ampure XP Beads</td>
			<td style="width:147px;">Beckman Coulter</td>
			<td style="width:119px;">A63880</td>
			<td style="width:369px;">Referred to in the text as solid phase reversible immobilization (SPRI) beads</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">DNA Gel Loading Dye (6x)</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">R0611</td>
			<td style="width:369px;">Referred to in the text as 6X loading dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Ethyl alcohol</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">E7023-500ML</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">KAPA SYBR FAST One-Step qRT-PCR Kits</td>
			<td style="width:147px;">Kapa BioSystems</td>
			<td style="width:119px;">7959613001</td>
			<td style="width:369px;">Referred to in the text as 2X qPCR mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">N,N&#8242;-Methylenebis(acrylamide)&#160;</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">146072-100G</td>
			<td style="width:369px;">Also known as bis-acrylamide</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:271px;">NexteraXT DNA Library Preparation Kit (referred to in the text as library preparation kit)</td>
			<td style="width:147px;">Illumina</td>
			<td style="width:119px;">FC-131-1024</td>
			<td style="width:369px;">Includes: TD (referred to in the text as transposase buffer), ATM (referred to in the text as transposase), NT (referred to in the text as transposase stop buffer), and NPM (Referred to in the text as library prep PCR mix)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Nuclease Free Water</td>
			<td style="width:147px;">Millipore</td>
			<td style="width:119px;">3098</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Protease</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:119px;">19155</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="189">
			<td height="189" style="height:189px;width:271px;">SMART-Seq v4 Kit (referred to in the text as whole-transcript amplification (WTA) kit)</td>
			<td style="width:147px;">Takara/Clontech</td>
			<td style="width:119px;">634888</td>
			<td style="width:369px;">Includes: Lysis buffer, RNase inhibitor, 3&#8217; SMART-Seq CDS Primer II A (referred to in the text as RT primer), 5X Ultra Low First Strand Buffer (referred to in the text as first strand buffer), SMART-Seq v4 Oligonucleotide (referred to in the text as template switch oligonucleotide (TSO)), SMART-Scribe Reverse Transcriptase (referred to in the text as reverse transcriptase), and PCR Primer II A (referred to in the text as cDNA PCR primer)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Random hexamer with WTA adapter&#160;</td>
			<td style="width:147px;">IDT</td>
			<td style="width:119px;">n/a</td>
			<td>5&#8242;-AAGCAGTGGTATCAACGCAGAGTAC-NNNNNN-3&#8242;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Sucrose</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">S0389-500G</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">TEMED</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">T9281-25ML</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">DNA AWAY Surface Decontaminant</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">7010PK&#160;&#160;</td>
			<td style="width:369px;">Referred to in the text as DNA removal product</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Tris-Borate-EDTA buffer (10X concentration)</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">T4415-1L</td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">SYBR Gold Nucleic Acid Gel Stain (10,000X Concentrate in DMSO)</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">S11494</td>
			<td style="width:369px;">Referred to in the text as gel stain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;"></td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Equipment</td>
			<td style="width:147px;"></td>
			<td style="width:119px;"></td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">BD Precisionglide&#160;syringe needles, gauge 18</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">Z192554</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Branson CPX series ultrasonic bath</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">Z769363</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Empty Gel Cassettes, mini, 1.0 mm</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">NC2010</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:271px;">Mesh Filter Plate - Corning&#160;HTS Transwell 96 well permeable supports - 8.0 &#181;m pore size</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">CLS3374</td>
			<td style="width:369px;">Referred to in the text as 8 um mesh filter plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">PowerPac HC Power Supply</td>
			<td style="width:147px;">Bio-Rad</td>
			<td style="width:119px;">1645052</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Qubit Fluorometer</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">Q33216</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Vacufuge Concentrator</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:119px;">22822993</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">XCell SureLock Mini-Cell system</td>
			<td style="width:147px;">ThermoFisher Scientific</td>
			<td style="width:119px;">EI0001</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Bio-Rad CFX96 Touch Real-Time PCR Detection System</td>
			<td style="width:147px;">Bio-Rad</td>
			<td style="width:119px;">1855195&#160;</td>
			<td style="width:369px;">Any equivalent hardware is acceptable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Amersham UVC 500 Ultraviolet Crosslinker</td>
			<td style="width:147px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">UVC500-115V</td>
			<td style="width:369px;">Discontinued, any equivalent hardware is acceptable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">Gel Doc XR+ Gel Documentation System</td>
			<td style="width:147px;">Bio-Rad</td>
			<td style="width:119px;">1708195</td>
			<td style="width:369px;">Referred to in the text as gel imager</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:271px;">Dark Reader Transilluminator</td>
			<td style="width:147px;">Clare Chemical Research</td>
			<td style="width:119px;">DR89</td>
			<td style="width:369px;">Referred to in the text as UV transilluminator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:271px;">&#160;Ultrasonic&#160;Bath</td>
			<td style="width:147px;">Bransonic</td>
			<td style="width:119px;">1207K35</td>
			<td style="width:369px;">Any equivalent ultrasonic bath is acceptable.</td>
		</tr>
	</tbody>
</table>

gel-seq: a method for simultaneous sequencing library preparation of dna and rna using hydrogel matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

The physical separation of gDNA and cDNA/RNA hybrids in the Gel-seq device can be visualized through fluorescent gel imaging; a representative result is shown in Figure 3. Panel A shows the fabricated Gel-seq device; false color has been added to distinguish the different gel regions. Panel B shows a close up of four different separations used for validation. The third lane, a negative control, represents background and shows that there is no autoflourescence of the gel at the interfaces. We loaded the first and second lanes with DNA ladders. These lanes show only a dark band at the interface between the low- and high-density membranes, revealing that small fragments can pass through the low-density gel. The fourth lane shows the behavior of a biological sample of interest: 500 PC3 cells. We loaded lane four as described in step 3 of the protocol. The image shows separation of genomic DNA and cDNA/RNA hybrids. A dark band at the top of the low-density membrane is megabase-scale genomic DNA while the cDNA/RNA hybrids are stacked at the interface of the low and high-density regions. Unlike the lanes loaded with ladder, there are also several bands present within the high-density region of the gel. These fragments, smaller than 100 bp, are off-target products generated from primer oligonucleotides during reverse transcription. Panel C shows a representative image of the entire Gel-seq device from a successful experiment. Lanes labeled RNA/cDNA were processed with the Gel-seq protocol, while lanes labeled RNA show a separation of just gDNA and RNA. Panel D shows a failed experiment with black bands at the top of every lane of the low-density membrane. This was caused by electrophoresis buffer contaminated with fragmented DNA. At this step, researchers should be looking for both clean negative controls and two distinct black bands indicating the presence of separated gDNA and RNA/cDNA hybrids.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57315/57315fig3.jpg" /> 
Figure 3. Gel-Seq Separation Results.&#160;The Gel-seq device (A) and a fluorescent image showing separation of DNA and RNA/cDNA hybrids (B). False color has been added to more easily distinguish between the different regions of density within the gel. (C and D) Representative fluorescent images of the entire Gel-seq device from a successful (C) and failed (D) experiment. NTC = No Template Control. Portions of this figure were reproduced from Ref. 7 with permission from the Royal Society of Chemistry.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57315/57315fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Once the DNA and RNA/cDNA hybrids have been separated and the remainder of the Gel-seq protocol completed, it is possible to generate sequencing libraries. To validate the prepared libraries, we run either a standard gel electrophoresis experiment (Figure 4) or a bioanalyzer. The results in Figure 4 show libraries generated from 500 PC3 cells and 750 HeLa cells. The figure shows the fragment distributions for matched libraries generated from Gel-seq (labeled &#39;Gel&#39;) compared to unmatched samples generated with standard protocols (labeled &#39;Tube&#39;). The fragment sizes for Gel-seq appear between 200 and 800 basepairs as expected when preparing libraries using the standard whole-genome library preparation kit. If the library fragments do not appear in the correct size range in this step, library preparation has failed.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57315/57315fig4.jpg" /> 
Figure 4. Library Fragment Size Comparison.&#160;A fluorescent gel electrophoresis image comparing library size distribution between Gel-seq (Gel) and standard controls (Tube). The left lane contains a low mass DNA ladder with fragment sizes 100, 200, 400, 800, 1200, and 2000 basepairs. The fragment sizes for all libraries fall between 200 and 800 basepairs, as expected for libraries prepared with this kit.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57315/57315fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The ultimate validation of the Gel-seq protocol is based on the analysis of sequencing results. We selected PC3 cells for our validation experiments, as these cells have homogenous expression profiles that allow samples to be split and processed using both Gel-seq and traditional methods; see Figure 5. A comparison between genomic DNA for PC3 cells is shown in Figures 5A and 5B. Figure 5A shows a comparison of genome-wide copy number variation (CNV) profiles generated from PC3 using either Gel-seq or a standard whole-genome library prep reaction (tube control). Each point is a mean normalized bin count; bins are defined from reference genome data such that each bin has equal expected count in a healthy diploid cell, i.e., a flat line, representing equal copies for each region of all autosomal (excluding X and Y) chromosomes. PC3 contains multiple copies of the same regions which show up as spikes above a background copy number of two. Gel-seq yields a qualitatively similar CNV profile as standard tube reaction. Agreement between the two plots can be assessed quantitatively by linear regression, as shown in panel B. A Pearson correlation of R = 0.90 indicates that genomic data gathered from either method is functionally equivalent.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57315/57315fig5.jpg" /> 
Figure 5. Library Validation.&#160;Gel-seq validation for the genomic (A and B) and transcriptomic (C, D, and E) data generated from PC3 and Hela cells. Panel A shows a comparison of genome-wide CNV profiles generated from PC3 using either Gel-seq (Gel-seq) or a standard reaction (tube). MAPD = Median Absolute Pairwise Difference. Panel B is a linear regression between the two samples in Panel A, with R = 0.90 indicating the genomic data are functionally equivalent. The axes show the log2 normalized bin counts. Panels C and D compare transcriptomic data from PC3 cells, with each point showing a count in transcripts per kilobase per million (TPM). The axes show the comparison between two samples as log2 normalized transcript counts. Panel C shows a comparison of technical replicates generated using Gel-seq, and Panel D shows a comparison between Gel-seq and traditional RNA-seq. Panel E shows that Gel-seq can resolve cell type based on RNA expression using a principal component analysis. The x axis shows that the first principal component accounts for 91.6% of the variation between the samples while the y axis shows that the second principal component accounts for only 6.5% of the variance. Portions of this figure were reproduced from Ref. 7 with permission from the Royal Society of Chemistry.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57315/57315fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Similarly, we compared transcriptomic data from our Gel-seq protocol to the standard in-tube Smart-Seq protocol. Figure 5 shows the correlation between both Gel-seq technical replicates (Figure 5C) and between Gel-seq and the standard method (Figure 5D). Each point is a count in transcripts per kilobase per million (TPM) for each gene detected at TPM &#62; 5 in both datasets. The linear regressions are shown as red lines, and the Pearson correlation coefficient is shown in the upper left corner. Technical replicates from Gel-seq agree (R &#8764; 0.8), but correlate less well with the standard method (R &#60; 0.7). This suggests that Gel-seq introduces a bias in gene counts. Fortunately, this bias is systematic and, as can be seen by the principal component analysis in Figure 5E, meaningful conclusions can still be drawn between different biological samples.

Watch this Scientific Journal Video about Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices at JoVE.com

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...

gel-seq-method-for-simultaneous-sequencing-library-preparation-dna

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.9K Views.  University of San Diego. Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

Video: Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform to study protein-protein and protein-lipid interactions3, monitor drug delivery4,5, and encapsulation4. Phospholipids naturally create curved lipid bilayers, distinguishing itself from a micelle.6 Liposomes are traditionally classified by size and number of bilayers, i.e. large unilamellar vesicles (LUVs), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs)7. In particular, the preparation of homogeneous liposomes of various sizes is important for studying membrane curvature that plays a vital role in cell signaling, endo- and exocytosis, membrane fusion, and protein trafficking8. Several groups analyze how proteins are used to modulate processes that involve membrane curvature and thus prepare liposomes of diameters &lt;100 - 400 nm to study their behavior on cell functions3. Others focus on liposome-drug encapsulation, studying liposomes as vehicles to carry and deliver a drug of interest9. Drug encapsulation can be achieved as reported during liposome formation9. Our extrusion step should not affect the encapsulated drug for two reasons, i.e. (1) drug encapsulation should be achieved prior to this step and (2) liposomes should retain their natural biophysical stability, securely carrying the drug in the aqueous core. These research goals further suggest the need for an optimized method to design stable sub-micron lipid vesicles.
Nonetheless, the current liposome preparation technologies (sonication10, freeze-and-thaw10, sedimentation) do not allow preparation of liposomes with highly curved surface (i.e. diameter &lt;100 nm) with high consistency and efficiency10,5, which limits the biophysical studies of an emerging field of membrane curvature sensing. Herein, we present a robust preparation method for a variety of biologically relevant liposomes.
Manual extrusion using gas-tight syringes and polycarbonate membranes10,5 is a common practice but heterogeneity is often observed when using pore sizes &lt;100 nm due to due to variability of manual pressure applied. We employed a constant pressure-controlled extrusion apparatus to prepare synthetic liposomes whose diameters range between 30 and 400 nm. Dynamic light scattering (DLS)10, electron microscopy11 and nanoparticle tracking analysis (NTA)12 were used to quantify the liposome sizes as described in our protocol, with commercial polystyrene (PS) beads used as a calibration standard. A near linear correlation was observed between the employed pore sizes and the experimentally determined liposomes, indicating high fidelity of our pressure-controlled liposome preparation method. Further, we have shown that this lipid vesicle preparation method is generally applicable, independent of various liposome sizes. Lastly, we have also demonstrated in a time course study that these prepared liposomes were stable for up to 16 hours. A representative nano-sized liposome preparation protocol is demonstrated below.

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

Liposomes are artificially prepared vesicles consisting of natural and synthetic phospholipids that are widely used as a cell membrane mimicking platform ...

An Improved Method for the Preparation of Type I Collagen From Skin

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.

Traditional procedures for the isolation of soluble type 1 collagen (COL1) require about 10 days from start to finish because of lengthy buffer incubations and laborious resuspensions of fibrils. Here, we describe a means to purify COL1 from small dermal biopsies in less than 3 hr.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes and analysis of complex structural variants. Nanopore platforms perform single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores and can generate hundreds of kilobase (kb) reads with minimal capital cost. This platform has been adopted by many researchers for a variety of applications. Achieving longer sequencing read lengths is the most critical factor to leverage the value of nanopore sequencing platforms. To generate ultra-long reads, special consideration is required to avoid DNA breakages and gain efficiency to generate productive sequencing templates. Here, we provide the detailed protocol of ultra-long DNA sequencing including high molecular weight (HMW) DNA extraction from fresh or frozen cells, library construction by mechanical shearing or transposase fragmentation, and sequencing on a nanopore device. From 20-25 &#181;g of HMW DNA, the method can achieve N50 read length of 50-70 kb with mechanical shearing and N50 of 90-100 kb read length with transposase mediated fragmentation. The protocol can be applied to DNA extracted from mammalian cells to perform whole genome sequencing for the detection of structural variants and genome assembly. Additional improvements on the DNA extraction and enzymatic reactions will further increase the read length and expand its utility.

Long-read sequences greatly facilitate the assembly of complex genomes and characterization of structural variation. We describe a method to generate ultra-long sequences by nanopore-based sequencing platforms. The approach adopts an optimized DNA extraction followed by modified library preparations to generate hundreds of kilobase reads with moderate coverage from human cells.

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

High Throughput Yeast Strain Phenotyping with Droplet-Based RNA Sequencing

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.

A bottleneck in the &#8216;design-build-test&#8217; cycle of microbial engineering is the speed at which we can perform functional screens of strains. We describe a high-throughput method for strain screening applied to hundreds to thousands of yeast cells per experiment that utilizes droplet-based RNA sequencing.

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...