We would like to acknowledge Bettine Gibbs for her technical help.

Wear a lab coat, gloves, and safety glasses at all times while performing all procedures in this protocol. Ensure that containers/tubes containing the phenol and guanidine isothiocyanate reagent are only opened in a certified hood, and dispose of phenol waste in a designated container. Use DNAse/RNAse-free sterile tubes, tips, and reagents.
1. Cell Culture
<ol>
	<li>Grow the HeLa cell line and two suspension mantle cell lymphoma cell lines, Granta-519 and Jeko-1, in DMEM containing 10% FBS and 100 U/mL Penicillin/Streptomycin in a humidified incubator with 5% CO2 at 37 &#176;C. Dilute the cells 1:10 and count using a particle counter14.</li>
	<li>For RNA extraction, plate 500,000 cells/well in a 6 well plate (2 mL media per well). After incubation for 24 h, collect RNA from the cells.</li>
</ol>
2. RNA Extraction
<ol>
	<li>Harvesting cells 
	NOTE: With the exception of the centrifugation step, perform all the listed steps in a tissue culture hood to maintain sterility.
	<ol>
		<li>For suspension cells (Jeko-1 and Granta-519):
		<ol>
			<li>Transfer the cells (together with 2 mL of media) to a 15 mL tube, and centrifuge at 1,725 x g for 5 min. Aspirate the media and leave the cell pellet in the bottom of the tube.</li>
			<li>Resuspend the cell pellet in 50 &#181;L of 1x Phosphate Buffered Saline (PBS). Add 500 &#181;L of monophasic phenol and guanidine isothiocyanate reagent to each sample in the 1.5 mL microcentrifuge tube. Mix well and leave at room temperature (RT) for 5 min while inverting the microcentrifuge tube every 1 min.</li>
		</ol>
		</li>
		<li>For adherent cells (HeLa):
		<ol>
			<li>Aspirate the cell culture media from each well. Add 1 mL of PBS to each well to wash off debris.</li>
			<li>Remove the PBS and add 500 &#181;L of monophasic phenol and guanidine isothiocyanate reagent to each well.</li>
			<li>Incubate at RT for 5 min with gentle rocking. Transfer to a 1.5 mL microcentrifuge tube.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cell lysis
	<ol>
		<li>Freeze the phenol and guanidine isothiocyanate cell mixture at -20 &#176;C for 1 h. 
		NOTE: The mix can be left at -20 &#176;C overnight or until needed.</li>
		<li>Thaw the phenol and guanidine isothiocyanate cell mixture on ice. Add 100 &#181;L of chloroform to the microcentrifuge tube in a PCR hood. Vortex the sample for 10 - 15 s until the mixture is pink and opaque. Incubate the sample on ice (at 4 &#176;C) for 15 min.</li>
		<li>Centrifuge the sample for 15 min at 20,800 x g and 4 &#176;C. Carefully remove the upper colorless aqueous phase (~ 200 &#181;L) and transfer to a new 1.5 mL microcentrifuge tube.</li>
	</ol>
	</li>
	<li>RNA precipitation
	<ol>
		<li>Add an equal volume of isopropanol to each sample. Add 1 &#181;L of coprecipitant (see Table of Materials) to each sample to act as a carrier for the RNA and help visualize the RNA in subsequent steps. Incubate the sample at -80 &#176;C for at least 4 h. For best results, incubate overnight at -80 &#176;C.</li>
		<li>Transfer the sample to a cooled centrifuge. Centrifuge for 35 min at 20,800 x g / 4 &#176;C; the RNA will appear as a blue speck on the bottom of the tube. Carefully remove the isopropanol from the sample. Add 500 &#181;L of 80% ethanol and resuspend the pellet by briefly vortexing for 3 s.</li>
		<li>Centrifuge the sample at 20,800 x g at RT for 5 min. Remove all the ethanol from the sample. Let the sample air-dry for about 10 min. Resuspend the sample in 35 &#181;L of RNAse-free water. Place on a heat block (65 &#176;C) for 5 min to ensure that the RNA is fully in solution, and immediately place the tube on ice.</li>
		<li>Determine the concentration of total RNA using a spectrophotometer as previously described, except use 1.5 &#181;L of the RNA sample instead of 1 &#181;L15. Optionally, run 1 &#181;g of total RNA on a 1% agarose gel to determine the RNA integrity.</li>
	</ol>
	</li>
</ol>
3. DNase Treatment
<ol>
	<li>After quantification of the RNA, perform DNAse treatment on the total RNA to degrade any genomic DNA. For each sample, add the following reagents from the RNAse-free DNAse kit to make a 20 &#181;L total reaction mix in a separate microcentrifuge tube:
	<ol>
		<li>Mix 2 &#181;L of DNase 10X reaction buffer with 4.4 &#181;g of total RNA. Bring to a total of 14 &#181;L with water.</li>
		<li>Add 2 &#181;L of RNase-free DNAse. Incubate at 37 &#176;C for 30 min in the presence of 2 &#181;L of RNase inhibitor (from the Reverse Transcription Kit).</li>
		<li>Add 2 &#181;L of Stop Solution and heat on a heat block for 10 min at 70 &#176;C to inactivate the DNAse enzyme. 
		NOTE: DNAse treatment is optional.</li>
	</ol>
	</li>
</ol>
4. cDNA Synthesis
For a final reaction volume of 50 &#181;L:
<ol>
	<li>For a final reaction volume of 50 &#181;L: Transfer 22 &#181;L of the DNase treated RNA to a new tube, or transfer 4 &#181;g of total RNA with added water to a total volume of 22 &#181;L to a new tube. Add 2 &#181;L of the T7 oligo dT25 primer from 10 &#181;M primer solution. The sequence for the T7 oligo dT25 primer is 5&#39;-GCCGGTAATACGACTCACTATAGTTTTTTTTTTTTTTTTTTTTTTTTT-3&#39;.</li>
	<li>From the Reverse Transcription kit add 2 &#181;L of the RNase inhibitor (20 U/&#181;L), 8 &#181;L of the 5x&#160;Reaction buffer, 4 &#181;L of 10 mM dNTPs, and 2 &#181;L of the Reverse transcriptase (200 U/&#181;L). 
	NOTE: Set up a reaction without reverse transcriptase to act as a negative control.</li>
	<li>Incubate at 42 &#176;C for 1 h. Transfer directly to ice. Heat the tube at 75 &#176;C for 5 min and place the tube on ice.</li>
</ol>
5. Primer Search for Fusion Gene Transcript
<ol>
	<li>Primer design
	<ol>
		<li>Download the cDNA sequence from the Ensemble genome browser (ENST00000360839.6/NM_017747), and identify a region within the ORF of the target transcript (ANKHD1) upstream of the stop codon. Copy and paste the entire region (up to 700 nucleotides) containing sequences upstream of the stop codon into the sequence entry region of the primer design software (&#60;https://www.idtdna.com/Primerquest/Home/Index&#62;).</li>
		<li>Select the show custom design option and select 10 for the number of primers that the system should generate (results to return) and leave all the other parameters set to default.</li>
		<li>Choose five forward primers and two reverse primers that do not overlap. Order primers and reconstitute with RNAse/DNAse-free water to a final concentration of 10 &#181;M. More details about general primer design for PCR are covered elsewhere16.</li>
	</ol>
	</li>
	<li>PCR to identify potential primers for use in subsequent 3&#39; RACE 
	NOTE: To determine the final forward primers to use in the reaction, test the designed primers by setting up different combinations of forward and reverse primers for regular PCR in a PCR hood.
	<ol>
		<li>Transfer the cDNA (2 &#181;L) to a fresh 0.5 mL PCR tube. Add 1 &#181;L of the forward primer and 1 &#181;L of the reverse primer (from a 10 mM primer solution). Make up to 12.5 &#181;L by adding 8.5 &#181;L of nuclease-free water. Add 12.5 &#181;L of the 2x PCR-to-Gel Master Mix.</li>
		<li>Use the following PCR thermal cycling conditions: 95 &#176;C for 2 min followed by 29 cycles of 95 &#176;C for 30 s, 60 &#176;C for 30 s, and 72 &#176;C for 1 min. Set the final cycle at 68 &#176;C for 7 min.</li>
		<li>After the PCR, run the products on a 1% agarose gel stained with ethidium bromide. Details of electrophoresis are as previously described with a few modifications17.
		<ol>
			<li>Dissolve 1 g of agarose in 100 mL of Tris-Acetate (TAE) buffer in a 250 mL flask. Boil in a microwave.</li>
			<li>Leave to cool for 1 min and add 7.5 &#181;L of ethidium bromide solution (10 mg/mL stock) in a fume hood. Mix well by swirling the flask and pour into a horizontal gel apparatus. Leave at RT in the fume hood for at least 30 min to allow the gel to solidify.</li>
			<li>Cover the gel with 1x&#160;TAE buffer and load 10 &#181;L of the PCR product onto the agarose gel together with 3 - 5 &#181;L of the DNA molecular weight ladder. Run at 175 V for 10 min. Visualize the products using an imager, and if further separation of products is needed, run the gel for an additional 5 - 10 min.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Selection of nested primers for 3&#39; RACE
	<ol>
		<li>Analyze the results of the PCR agarose gel to select primers that have a distinct, strong, single PCR band. Use the 5&#39;-most primer (ANKHD15&#39;-CCTTCCCAGCGAGTTTCTAC-3&#39;) together with the T7 oligodT25 primer in the first PCR run.</li>
		<li>Select the nested primer that is located downstream of the first primer (ANKHD1 5&#39;-CGTTGGACACAGTGGAATCT-3&#39;), and use it with the T7 primer (5&#39;-GGCCTAATACGACTCACTATAG-3&#39;) in the second set of the PCR reactions.</li>
	</ol>
	</li>
</ol>
6. Optimizing 3&#39; RACE to Map the 3&#39; UTR Using Two Different Enzymes
NOTE: There has been an increase in the diversity of DNA polymerases used for PCR; therefore, we wanted to determine standard conditions that can be applied even when using different enzymes for 3&#39; RACE PCR reactions. The reverse primers for any transcript are kept constant; the only changes are in the nested forward primers that are specific for the target transcript.
<ol>
	<li>Protocol 1: 3&#39; RACE using a modified DNA polymerase from Pyrococcus furiosus (Pfu)
	<ol>
		<li>First PCR
		<ol>
			<li>Transfer 1 &#181;L of cDNA to a PCR tube and add 5 &#181;L of 10x&#160;Pfu reaction buffer. Add 1 &#181;L of the first nested forward primer, 1 &#181;L of the T7 oligodT25 primer, and 1 &#181;L of dNTPs (from a 10 mM solution).</li>
			<li>Make up to 49 &#181;L total volume by adding 40 &#181;L of water. Add 1 &#181;L of Pfu DNA polymerase and mix well. Run the PCR using the PCR profile in Table 1.</li>
		</ol>
		</li>
		<li>Second PCR
		<ol>
			<li>Transfer 2 &#181;L of the products from the first PCR to a new PCR tube and mix with 5 &#181;L of 10x&#160;PfuUltra II reaction buffer. Add 1 &#181;L of the second nested PCR primer, 1 &#181;L of the T7 primer, and 1 &#181;L of dNTPs (10 mM solution).</li>
			<li>Make up to 49 &#181;L total reaction volume by adding 39 &#181;L of water. Add 1 &#181;L of Pfu DNA polymerase. Run the PCR using the same PCR profile as the first PCR setup (Table 1).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Protocol 2: 3&#39; RACE using a chimeric DNA polymerase consisting of a DNA binding domain fused to a Pyrococcus-like proofreading polymerase PCR master mix
	<ol>
		<li>First PCR
		<ol>
			<li>Transfer 1 &#181;L of cDNA to a PCR tube. Add 1 &#181;L of the first nested forward primer and 1 &#181;L of the T7 oligodT25 primer. Make up to 25 &#181;L by adding 22 &#181;L of water.</li>
			<li>Add 25 &#181;L of 2x&#160;PCR Master Mix and mix well. Use the PCR cycling conditions described in Table 2.</li>
		</ol>
		</li>
		<li>Second PCR
		<ol>
			<li>Transfer 2 &#181;L of the products from the first PCR to a new PCR tube. Add 1 &#181;L of the second nested PCR primer together with 1 &#181;L of the T7 reverse primer.</li>
			<li>Make up to 25 &#181;L by adding 22 &#181;L of water. Add 25 &#181;L of 2X PCR Master Mix. Run the PCR using the same PCR profile as the first PCR setup (Table 2).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
7. Verify the second PCR product of 3&#39; RACE.
<ol>
	<li>Take 5 - 10 &#181;L of the second PCR product (as well as product from the first round of PCRs if the transcript is abundantly expressed) and run on an agarose gel. Set up the gel and run the electrophoresis as previously described (steps 5.2.3.1 to 5.2.3.3). Visualize results on an imager.</li>
</ol>
8. Product Purification and Sequencing
<ol>
	<li>Purify the gel PCR products from the second PCR using the Gel and PCR Fragment DNA Clean-Up System as per the manufacturer&#39;s protocol.</li>
	<li>Perform Sanger sequencing (alternatively, send the gel purified PCR product samples and appropriate sequencing primers to a sequencing lab). Analyze the sequencing data after Sanger sequencing.</li>
	<li>Download sequence-analyzing software (see Table of Materials) and import the trace files into the software. Use the individual PURE Base QVs (Quality Values) to obtain the Base call information18. Download the chromatogram with high quality traces for viewing. 
	Optional: The gel purified product can be cloned using an appropriate cloning kit and the isolated clones sent for Sanger sequencing.</li>
</ol>

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The authors have nothing to disclose.

Despite the advent of massive parallel sequencing technologies, on a gene-by-gene basis, 3&#39; RACE still remains the easiest and most economical method to identify the PAS and nucleotides adjacent to the poly(A) tail. The adaptation described here expands using 3&#39; RACE to both amplify and map sequences that include a portion of the ORF, the stop codon, and the entire 3&#39; UTR of the ANKHD1 mRNA transcript. A major advantage of 3&#39; RACE is that with a few minor adaptations, products from 3&#39; RACE can be cloned into other vectors to facilitate downstream interrogation of 3&#39; UTR function including miRNA targeting, stability assays as well as other mechanistic assays. This can be done by including restriction enzyme sites within the nested primer sequences9,10. Adjustments can be made to include only the 3&#39; UTR without any sequences from the ORF for cloning for 3&#39; UTR functional assays5.
A major determinant of the success of 3&#39; RACE is the development of nested primers that target the gene of interest. The cDNA sequences from the Ensembl genome browser are recommended to best identify regions without polymorphisms in order to develop optimal primers. Ideally, several nested forward primers as well as at least two reverse primers within the gene of interest can be screened using standard PCR to identify the best two nested gene specific primers to use. In this study the first gene specific forward primer selected for 3&#39; RACE initially gave two bands with standard PCR routinely used in the lab. Unfortunately, the primer design was limited by the significant number of SNPs in the transcript of interest. SNPs in the target transcript lead to mismatch between the primers and the target, which can result in inefficient annealing and amplification, and should be reduced to a minimum19. The presence of at least four SNPs in one primer, or the occurrence of a combination of five mismatches (three in one primer and two in the other) may result in complete inhibition of the PCR reaction, and hence the only option is to design more primers20. Instead of designing more primers to get one single band for the standard PCR used for the primer search in experiments reported here, the PCR cycling conditions and the DNA polymerases subsequently used in 3&#39; RACE were altered in order to optimize the probability of getting one specific band. The denaturation temperature was increased to 98 &#176;C for one of the DNA polymerases (an increase from 95 &#176;C used in the primer search). For both DNA polymerases used in the 3&#39; RACE PCR, the initial denaturation duration was increased to 3 min instead of the recommended 30 s to 2 min in order to fully denature the cDNA template. The T7 oligo dT25 primer for example is predicted to potentially form weak secondary structures, which may reduce the availability of primers21. Increasing the initial denaturation length and temperature breaks any secondary structures and helps yield the single specific band after the final cycle of 3&#39; RACE. In addition, the non-specific band was lighter than the expected bands, suggesting that it appeared at higher PCR cycles (up to 35 cycles); limiting the PCR cycles to only 20 reduces the amplification of the non-specific band.
Another potential of 3&#39; RACE, like other PCR based reactions, is incomplete amplification of the target region22. Hence, for both thermophilic enzymes the initial annealing temperature was set at 50 &#176;C for 5 min to optimize annealing of the primers to the target and this was followed by an initial extension temperature at 72 &#176;C for 10 min. The final PCR cycle for each enzyme had a final extension time of 15 min at 72 &#176;C. These extension steps were longer than those recommended by the manufacturers of the DNA polymerases. The long extension (elongation) step allows complete synthesis of incomplete amplicons, enabling full extension of the initial and final amplification products16.
PCR steps that occur in 3&#39; RACE are highly sensitive, so they may detect genomic DNA (or other DNA contamination) instead of the target mRNA transcript, resulting in several unexpected bands on the gel22. One way to prevent contamination is to perform the PCR work in a dedicated PCR hood, wear gloves, and use clean reagents and autoclaved sterile tubes and pipette tips. In regular PCR, forward and reverse primers can be designed so they are located on different exons (across intron primers); in this way, an abnormally large PCR product would signify that a region containing an intron has been amplified. However, this may not always be feasible in the event that only the 3&#39; UTR sequence, located within the same terminal exon, is the target for 3&#39; RACE amplification. Alternatively, the RNA can be pre-treated with DNAse enzyme before cDNA synthesis. The use of the T7 oligo dT25 primer to prime the reverse transcriptase reaction instead of a random hexanucleotide for first strand cDNA synthesis from RNA also decreases amplification of the genomic products. Last but not least, setting up a negative control (the &#34;&#8211;&#34; lanes in Figure 2), where cDNA synthesis is set up in absence of reverse transcriptase is highly recommended. The control undergoes identical steps to the other samples in the subsequent 3&#39; RACE procedures. The presence of a band in this control signifies potential genomic DNA contamination.
Despite the aforementioned challenges, 3&#39; RACE is a powerful tool in mapping 3&#39; ends on an individual gene basis. The advent of next-generation technology has led to an increase in the repertoire of transcripts that have alternative 3&#39; ends resulting from alternative polyadenylation, which may or may not involve alternative splicing. To add to the complexity, in cancer cells, chromosomal rearrangements are frequent and can result in oncogenic gene fusion products23. The fusion between the two genes may involve the ORF or, in some cases, involve only sequences within the 3&#39; UTR9,10. Furthermore, there are also conjoined/co-transcribed genes, which are transcribed simultaneously but may be translated into different proteins11. 3&#39; RACE can be tailored to map all these unique, abnormal transcripts as well as normal transcripts. This is important because these abnormal transcripts may end up serving as disease specific biomarkers or specific drug targets, e.g., the drug Imatinib targets the BCR-ABL1 fusion gene in cancer.
Hence, 3&#39; RACE is a highly versatile technique which can be used to amplify normal transcripts, identify novel 3&#39; UTRs, and novel gene fusions within the 3&#39; UTR5,9. In this report, 3&#39; RACE was used to amplify and map the stop codon, the PAS, and the entire 3&#39; UTR of the ANKHD1 transcript using different DNA polymerases. The described approach can be used to map the 3&#39; end of any polyadenylated transcript.

The formation of the 3&#39; end is a critical step in mRNA maturation that comprises the cleavage of the pre-mRNA downstream of a PAS followed by the addition of ~250 untemplated adenines, which make up the poly(A) tail1,2. The poly(A) binding protein (PABP) binds to the poly(A) tail, and this protects the mRNA transcript from degradation, and facilitates translation1.
Current estimates suggest that 70% of human genes have multiple PASs, and thus undergo alternative polyadenylation, resulting in multiple 3&#39; ends3. Thus, it is important to identify where the poly(A) tail attaches to the rest of the 3&#39; UTR, as well as identify the PAS used by any given transcript. The advent of next-generation sequencing has resulted in the simultaneous identification of the 3&#39; UTRs and the PASs of thousands of genes. This increase in sequencing capability has required the development of bioinformatic algorithms to analyze data involving alternative polyadenylation of the 3&#39; end. For the de novo detection or validation of the PAS and hence mapping of the 3&#39; end of individual genes from large scale sequencing data, 3&#39; RACE remains the method of choice4,5. The sequences included in cDNA products of 3&#39; RACE normally include only a portion of the 3&#39; UTR that contains the poly(A) tail, the cleavage site, the PAS, and the sequences upstream of the PAS. Unlike PCR, which requires the design and use of gene specific forward and reverse primers, 3&#39; RACE only requires two gene specific nested forward primers. Hence, PCR requires a more detailed knowledge of the nucleotide sequence of a large region of the gene being amplified4,6. Since 3&#39; RACE uses the same reverse primer that targets the poly(A) tail for all polyadenylated RNA transcripts, only the forward primers need to be gene specific, thus, only requiring knowledge of a significantly smaller region of the mRNA. This enables the amplification of regions whose sequences are not fully characterized4,7. This has allowed 3&#39; RACE to be used not only to determine the 3&#39; end of a gene, but to also determine and characterize large regions upstream of the PAS that form a significant portion of the 3&#39; UTR. By combining 5&#39; RACE with the modified 3&#39; RACE that includes larger portions of the 3&#39; UTR and flanking regions, it is possible to fully sequence or clone an entire mRNA transcript from the 5&#39; end to its 3&#39; end8.
An example of this application of modified 3&#39; RACE is the recent identification of a novel CCND1-MRCK fusion gene transcript from Mantle Cell Lymphoma cell lines and cancer patients. The 3&#39; UTR consisted of sequences from both the CCND1 and MRCK genes and was recalcitrant to miRNA regulation9. The two nested CCND1 specific forward primers were complementary to the region immediately adjacent and downstream of the CCND1 stop codon. Although whole transcriptome sequencing together with specific bioinformatic tools can be used to detect gene fusions within the 3&#39; UTR10, many labs may lack the financial resources or bioinformatic expertise to make use of this technology. Hence, 3&#39; RACE is an alternative for de novo identification and validation of novel fusion genes involving the 3&#39; UTR. Considering the drastic increase in the number of reported fusion genes as well as read through transcripts, 3&#39; RACE has become a powerful tool in characterizing gene sequences11,12. In addition, recent studies have shown that different sequences within the 3&#39; UTR as well as the length of the 3&#39; UTR can affect mRNA transcript stability, localization, translatability, and function13. Due in part to an increased interest in mapping the transcriptome, there has been an increase in the number of different DNA polymerases being developed for use in the lab. It is important to determine what types of modifications can be made to the 3&#39; RACE protocol in order to utilize the available repertoire of DNA polymerases.
This work reports adapting 3&#39; RACE to map the entire 3&#39; UTR, the PAS, and the 3&#39; end cleavage site of the ANKHD1 transcript by using nested primers within the ANKHD1 section of the transcript and&#160;two different DNA polymerases.

<table><tbody><tr><td>HeLa cells</td><td>ATCC</td><td>CCL-2</td><td>Cervical cancer cell line.</td></tr><tr><td>Jeko-1 cells</td><td>ATCC</td><td>CRL-3006</td><td>Mantle cell lymphoma cell line.</td></tr><tr><td>Granta-519 cells</td><td>DSMZ</td><td>ACC-342</td><td>Mantle cell lymphoma cell line.</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma Aldrich</td><td>F6178</td><td>Fetal bovine serum for cell culture.</td></tr><tr><td>Penicillin/Streptomycin</td><td>ThermoFisherScientific</td><td>15140122</td><td>Antibiotic and antimycotic.</td></tr><tr><td>GlycoBlue</td><td>Ambion</td><td>AM9545</td><td>Coprecipitant.</td></tr><tr><td>DMEM</td><td>ThermoFisherScientific</td><td>10569044</td><td>Gibco brand cell culture media with GlutaMAX.</td></tr><tr><td>Nuclease Free-Water</td><td>ThermoFisherScientific</td><td>AM9938</td><td>Ambion DNase and RNAse free water(non DEPC treated).</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-Buffered Solution</td><td>Corning</td><td>21-030</td><td>1X PBS.</td></tr><tr><td>Chloroform</td><td>Sigma&amp;nbsp; Aldrich</td><td>C7559-5VL</td><td></td></tr><tr><td>2-propanol</td><td>Sigma Aldrich</td><td>I9516</td><td></td></tr><tr><td>Reagent Alcohol</td><td>Sigma Aldrich</td><td>793175</td><td>Ethanol</td></tr><tr><td>Ethidium Bromide solution</td><td>Sigma Aldrich</td><td>E1510</td><td></td></tr><tr><td>TRIzol Reagent</td><td>ThermoFisherScientific</td><td>15596026</td><td>Monophasic phenol and guanidine isothiocyanate reagent.</td></tr><tr><td>2X Extender PCR-to-Gel Master Mix</td><td>Amresco</td><td>N867</td><td>2X PCR-to-Gel Master Mix&amp;nbsp; containing loading dye used in routine quick PCR assays and primer search.</td></tr><tr><td>10mM dNTP</td><td>Amresco</td><td>N557</td><td></td></tr><tr><td>RQ1 RNase-Free DNase</td><td>Promega</td><td>M6101</td><td>Dnase treatment kit.</td></tr><tr><td>Gel Loading Dye Orange (6X)</td><td>New England BioLabs</td><td>B7022S</td><td></td></tr><tr><td>2X Phusion High-Fidelity PCR Master Mix with HF buffer</td><td>ThermoFisherScientific</td><td>F531S</td><td>2X PCR MasterMix containing a chimeric DNA polymerase consisting of a DNA binding domain fused to a Pyrococcus-like proofreading polymerase and other reagents.</td></tr><tr><td>PfuUltra II Fusion HS DNA polymerase</td><td>Agilent Technologies</td><td>600670</td><td>Modified DNA Polymerase from Pyrococcus furiosus (Pfu).</td></tr><tr><td>RevertAid RT Reverse Transcription Kit</td><td>Thermo Fischer</td><td>K1691</td><td>Used for&amp;nbsp; reverse transcription of mRNA into&amp;nbsp; cDNA synthesis. Kit includes&amp;nbsp; Ribolock RNAse inhibitor, RevertAid M-MuLV reverse transcriptase and other reagents listed in manuscript.</td></tr><tr><td>Pefect size 1Kb ladder</td><td>5 Prime</td><td>2500360</td><td>Molecular weight DNA ladder.</td></tr><tr><td>Alpha Innotech FluorChem Q MultiImage III</td><td>Alpha Innotech</td><td></td><td>Used to visualise ethidium bromide stained agarose gel.</td></tr><tr><td>Low Molecular Weight Ladder</td><td>New England BioLabs</td><td>N3233L</td><td>Molecular weight DNA ladder.</td></tr><tr><td>Vortex Mixer</td><td>MidSci</td><td>VM-3200</td><td></td></tr><tr><td>Mini Centrifuge</td><td>MidSci</td><td>C1008-R</td><td></td></tr><tr><td>Dry Bath</td><td>MidSci</td><td>DB-D1</td><td></td></tr><tr><td>NanoDrop 2000C</td><td>ThermoFisherScientific</td><td>ND-2000C</td><td>Spectrophotometer.</td></tr><tr><td>Wide Mini-Sub&amp;nbsp;Cell GT Horizontal Electrophoresis System</td><td>BioRad</td><td>1704469</td><td>Electrophoresis equipment-apparatus to set up gel</td></tr><tr><td>PowerPac Basic Power Supply</td><td>BioRad</td><td>1645050</td><td>Power supply for gel electrophoresis.</td></tr><tr><td>Agarose</td><td>Dot Scientific</td><td>AGLE-500</td><td></td></tr><tr><td>Mastercycler Gradient</td><td>Eppendorf</td><td>950000015</td><td>PCR thermocycler.</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810 R</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5430R</td><td></td></tr><tr><td>Wizard SV Gel and PCR Fragment DNA Clean-Up System</td><td>Promega</td><td>A9281</td><td></td></tr><tr><td>Zero Blunt TOPO PCR Cloning Kit, with One Shot&amp;nbsp; TOP10 Chemically Competent E. coli cells</td><td>ThermoFisherScientific</td><td>K280020</td><td></td></tr><tr><td>MyPCR Preparation Station Mystaire</td><td>MidSci</td><td>MY-PCR24</td><td>Hood dedicated to PCR work.</td></tr><tr><td>Hamilton SafeAire II fume hood</td><td>ThermoFisherScientific</td><td></td><td>Fume hood.</td></tr><tr><td>Beckman Coulter Z1&amp;nbsp;Particle&amp;nbsp;Counter</td><td>Beckman Coulter</td><td>6605698</td><td>Particle counter. For counting cells before plating&amp;nbsp; for RNA extraction.</td></tr><tr><td>Applied Biosystems Sequence Scanner Software v2.0</td><td>Applied Biosystems (through ThermoFisherScientific)</td><td></td><td>Software to analyze Sanger sequencing data.</td></tr></tbody></table>

adapting 3' rapid amplification of cdna ends to map transcripts in cancer

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the traditional method of choice is 3&#39; rapid amplification of cDNA ends (3&#39; RACE). Protocols for 3&#39; RACE require the careful design and selection of nested primers within the 3&#39; untranslated region (3&#39; UTR) of the target gene of interest. However, with a few modifications the protocol can be used to include the entire 3&#39; UTR and sequences within the open reading frame (ORF), providing a more comprehensive picture of the relationship between the ORF and the 3&#39; UTR. This is in addition to identification of the polyadenylation signal (PAS), as well as the cleavage and polyadenylation site provided by conventional 3&#39; RACE. Expanded 3&#39; RACE can detect unusual 3&#39; UTRs, including gene fusions within the 3&#39; UTR, and the sequence information can be used to predict potential miRNA binding sites as well as AU rich destabilizing elements that may affect the stability of the transcript.

Nested Forward Primer Search:
The agarose gel from Figure 1 shows two distinct PCR gel products (Lanes 1 and 2) which use the same forward primer but different reverse primers. Lane 3 has a distinct PCR product and has a distinct forward and reverse primer. The ideal primers to use for the PCR based reaction are those that give one distinct PCR product (Lane 3). The forward primer used in Lanes 1 and 2 gives the strongest band and gives the largest PCR products (expected), and is used as the first forward primer. The second nested primer for the second set of PCR reactions in 3&#39; RACE is the forward primer from Lane 3.
Different DNA Polymerases Used in 3&#39; RACE Produce Similar Results:
Two different DNA polymerases produced the same sized PCR products despite having different PCR cycling conditions for the 3&#39; RACE (Figure 2). There is only one distinct band for the expected PCR product. All of the minus reverse transcriptase negative controls (-) do not have a band, showing no genomic contamination of the RNA used in cDNA synthesis as well as in subsequent downstream reactions.
Sanger Sequencing Results:
The PCR products were gel purified and sent for Sanger sequencing. The results shown in Figure 3A show a portion of a sequence chromatograph from Sanger sequencing. A representative sequence identifies the location of the stop codon, putative polyadenylation signal, and the cleavage site, as well as the poly (A) tail in the 3&#39; RACE product (Figure 3B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57318/57318fig1.jpg" /> 
Figure 1: Identification of two nested gene specific forward primers to use in 3&#39; RACE. Shown is an example of PCR products from different combinations of gene specific forward and reverse primers using HeLa cDNA run on an ethidium bromide stained agarose gel with the molecular weight (mw) ladder. Lanes 1 and 2 are PCR products from the same gene specific&#160;forward primer but different reverse primers. The arrows point to the expected PCR product for each lane. In addition to the band for the expected PCR product, there is an additional band. The single PCR product in Lane 3 is from a different gene specific nested forward and reverse primer and shows the single expected band.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57318/57318fig2.jpg" /> 
Figure 2: Verification of 3&#39; RACE products from the second set of PCR reactions. Products from the second PCR reaction with (A) Pfu DNA Polymerase and (B) Chimeric DNA polymerase were run on an agarose gel with ethidium bromide. Lanes depicted by (-) are from the negative control sample for each cell line when cDNA synthesis from RNA was carried out in the absence of the reverse transcriptase enzyme. Lanes 1 and 2 are PCR products from biological replicates of each cell line. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57318/57318fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57318/57318fig3.jpg" /> 
Figure 3: Mapping the PCR products of 3&#39; RACE. (A) A portion of a sequence chromatogram of a gel purified 3&#39; RACE product showing the predicted polyadenylation signal and the location of the poly(A) tail. (B) Representative sequences from Sanger sequencing data showing the stop codon, the polyadenylation signal (PAS), the cleavage and attachment site of the poly(A) tail as well as a section of the poly (A) tail sequences. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57318/57318fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cycle step</td>
			<td>Temperature and Duration</td>
			<td>Number of cycles</td>
		</tr>
		<tr>
			<td>Initial denaturation</td>
			<td>95 &#176;C for 3 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Initial annealing and extension</td>
			<td>50 &#176;C for 5 min and 72 &#176;C for 10 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Subcyles (denaturation, annealing and extension)</td>
			<td>95 &#176;C for 40 s, 50 &#176;C for 1 min, and 72 &#176;C for 3 min</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Final cycle (denaturation, annealing and extension)</td>
			<td>95 &#176;C for 40 s, 50 &#176;C for 1 min, and 72 &#176;C for 15 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td>4 &#176;C, &#8734;</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Pfu DNA Polymerase PCR cycling conditions for 3&#39; RACE.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cycle step</td>
			<td>Temperature and Duration</td>
			<td>Number of cycles</td>
		</tr>
		<tr>
			<td>Initial denaturation</td>
			<td>98 &#176;C for 3 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Initial annealing and extension</td>
			<td>50 &#176;C for 5 min and 72 &#176;C for 10 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Subcyles (denaturation, annealing and extension)</td>
			<td>98 &#176;C for 30 s, 50 &#176;C for 1 min, and 72 &#176;C for 4 min</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Final cycle (denaturation, annealing and extension)</td>
			<td>98 &#176;C for 30 s, 50 &#176;C for 1 min, and 72 &#176;C for 15 min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Hold</td>
			<td>4 &#176;C, &#8734;</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Chimeric DNA polymerase PCR cycling conditions.

Watch this Scientific Journal Video about Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer at JoVE.com

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

The two different 3&#39; rapid amplification of cDNA ends (3&#39; RACE) protocols described here make use of two different DNA polymerases to map sequences that include a segment of the open reading frame (ORF), the stop codon, and the entire 3&#39; UTR of a transcript using RNA obtained from different cancer cell lines.

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the ...

adapting-3-rapid-amplification-cdna-ends-to-map-transcripts

Nanyang Technological University

Research

JoVE Journal

Genetics

12.3K Views.  Butler University. De twee verschillende 3' snelle versterking van cDNA uiteinden (3' RACE) protocollen beschreven hier maken gebruik van twee verschillende polymerase van DNA sequenties die bevatten een segment van het open leesraam (ORF), de stop codon en de gehele 3' UTR van een afschrift van het gebruik van RNA in kaart verkregen cellijnen van andere kanker.

Video: Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. However, the application of this method is challenging due to its low sensitivity in analyzing the trace amount of ctDNA in the blood. Furthermore, the method may generate false positive and negative results from this sequencing and subsequent analysis. To improve the feasibility and reliability of ctDNA detection in the clinic, here we present a technique which enriches rare mutations for sequencing, Enrich Rare Mutation Sequencing (ER-Seq). ER-Seq can distinguish a single mutation out of 1 x 107 wild-type nucleotides, which makes it a promising tool to detect extremely low frequency genetic alterations and thus will be very useful in studying disease heterogenicity. By virtue of the unique sequencing adapter&#39;s ligation, this method enables an efficient recovery of ctDNA molecules, while at the same time correcting for errors bidirectionally (sense and antisense). Our selection of 1021 kb probes enriches the measurement of target regions that cover over 95% of the tumor-related driver mutations in 12 tumors. This cost-effective and universal method enables a uniquely successful accumulation of genetic data. After efficiently filtering out background error, ER-seq can precisely detect rare mutations. Using a case study, we present a detailed protocol demonstrating probe design, library construction, and target DNA capture methodologies, while also including the data analysis workflow. The process to carry out this method typically takes 1-2 days.

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. ...

Cancer Research

Transcriptome Analysis of Single Cells

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in culture.1,2,3 In the case of the brain, regions such as the hippocampus contain a complex arrangement of different cell types, each with distinct mRNA profiles. The ability to harvest single cells allows for a more in depth investigation into the molecular differences between and within cell populations. We describe a simple and rapid method for harvesting cells for further processing. Pipettes often used in electrophysiology are utilized to isolate (using aspiration) a cell of interest and conveniently deposit it into an Eppendorf tube for further processing with any number of molecular biology techniques. Our protocol can be modified for the harvest of dendrites from cell culture or even individual cells from acute slices.
We also describe the aRNA amplification method as a major downstream application of single cell isolations. This method was developed previously by our lab as an alternative to other gene expression analysis techniques such as reverse-transcription or real-time polymerase chain reaction (PCR).4,5,6,7,8 This technique provides for linear amplification of the polyadenylated RNA beginning with only femtograms of material and resulting in microgram amounts of antisense RNA. The linearly amplified material provides a more accurate estimation than PCR exponential amplification of the relative abundance of components of the transcriptome of the isolated cell. The basic procedure consists of two rounds of amplification. Briefly, a T7 RNA polymerase promoter site is incorporated into double stranded cDNA created from the mRNA transcripts. An overnight in vitro transcription (IVT) reaction is then performed in which T7 RNA polymerase produces many antisense transcripts from the double stranded cDNA. The second round repeats this process but with some technical differences since the starting material is antisense RNA. It is standard to repeat the second round, resulting in three rounds of amplification. Often, the third round in vitro transcription reaction is performed using biotinylated nucleoside triphosphates so that the antisense RNA produced can be hybridized and detected on a microarray.7,8

In this article we describe a simple method for the harvesting of single cells from rat primary neuronal cultures and subsequent transcriptome analysis using aRNA amplification. This approach is generalizable to any cell type.

Many gene expression analysis techniques rely on material isolated from heterogeneous populations of cells from tissue homogenates or cells in ...

Neuroscience

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

ATAC-Seq Optimization for Cancer Epigenetics Research

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the hyperactive Tn5 transposase. Tn5 cuts and ligates adapters for high-throughput sequencing within accessible chromatin regions. In eukaryotic cells, genomic DNA is packaged into chromatin, a complex of DNA, histones, and other proteins, which acts as a physical barrier to the transcriptional machinery. In response to extrinsic signals, transcription factors recruit chromatin remodeling complexes to enable access to the transcriptional machinery for gene activation. Therefore, identifying open chromatin regions is useful when monitoring enhancer and gene promoter activities during biological events such as cancer progression. Since this protocol is easy to use and has a low cell input requirement, ATAC-seq has been widely adopted to define open chromatin regions in various cell types, including cancer cells. For successful data acquisition, several parameters need to be considered when preparing ATAC-seq libraries. Among them, the choice of cell lysis buffer, the titration of the Tn5 enzyme, and the starting volume of cells are crucial for ATAC-seq library preparation in cancer cells. Optimization is essential for generating high-quality data. Here, we provide a detailed description of the ATAC-seq optimization methods for epithelial cell types.

ATAC-seq is a DNA sequencing method that uses the hyperactive mutant transposase, Tn5, to map changes in chromatin accessibility mediated by transcription factors. ATAC-seq enables the discovery of the molecular mechanisms underlying phenotypic alterations in cancer cells. This protocol outlines optimization procedures for ATAC-seq in epithelial cell types, including cancer cells.

The assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) probes deoxyribonucleic acid (DNA) accessibility using the ...

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Biology

Oncogene Expression Analysis with Alterations in pH in a Pancreatic Ductal Cell Line

The fourth leading cause of cancer-related death, pancreatic ductal adenocarcinoma (PDAC), has a 12% five-year survival rate. This disease has a poor prognosis and is characterized by a rigid stromal microenvironment, which represents a tangible challenge in its treatment. Chronic pancreatitis patients have a 10-fold greater risk of developing PDAC; in these patients, the ductal pH decreases from pH 8.0 to pH 6.0 due to bicarbonate insufficiency and the inflammatory milieu. Our goal was to understand the role of the acidic environment observed in chronic pancreatitis on oncogene expression in a pancreatic ductal cell line.
Therefore, 80% confluent human pancreatic ductal epithelial cells were incubated at pH 6.0 to pH 7.2 for 6 h. Total RNA from the cells was processed to enrich the total mRNA in the samples. Gene expression was evaluated via next-generation sequencing (NGS) of biological replicates. RNA-seq analysis was carried out with the aid of an online tool, and the differentially expressed genes (FCs &#60; &#177; 2.0) were identified; there were 90, 148, and 109 upregulated genes and 20, 14, and 23 downregulated genes at pH 6.0, 6.5, and 7.0, respectively. Four oncogenes were upregulated at pH 6.0, seven were upregulated at pH 6.5, and seven were upregulated at pH 7.0. The common genes that were upregulated at pH 6.0, pH 6.5, and pH 7.0 were lymphocyte cell-specific protein-tyrosine kinase (LCK) [pH 6.0, FC: 2.93; pH 6.5, FC: 2.93; pH 7.0, FC: 3.32], FGR proto-oncogene, Src family tyrosine kinase (FGR) [pH 6.0, FC: 4.17; pH 6.5, FC: 5.25; pH 7.0, FC: 5.09], and ArfGAP With SH3 domain, ankyrin repeat, and PH domain 3 (ASAP3) [pH 6.0, FC: 2.37; pH 6.5, FC: 3.84; pH 7.0, FC: 2.51]. The acidic environment triggers the activation of proto-oncogenes, which may trigger tumor initiation in chronic pancreatitis.

This study describes a protocol for exploring the effects of altered pH on oncogene expression via RNA-seq analysis of a pancreatic ductal cell line.

The fourth leading cause of cancer-related death, pancreatic ductal adenocarcinoma (PDAC), has a 12% five-year survival rate. This disease has a poor ...

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

Medicine

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

3' End Sequencing Library Preparation with A-seq2

Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3&#39; untranslated regions (UTRs) are generated in differentiated cells whereas proliferating cells preferentially express transcripts with short 3&#39;UTRs. We describe the A-seq protocol, now at its second version, which was developed to map polyadenylation sites genome-wide and study the regulation of pre-mRNA 3&#39; end processing. Also this current protocol takes advantage of the polyadenylate (poly(A)) tails that are added during the biogenesis of most mammalian mRNAs to enrich for fully processed mRNAs. A DNA adaptor with deoxyuracil at its fourth position allows the precise processing of mRNA 3&#39; end fragments for sequencing. Not including the cell culture and the overnight ligations, the protocol requires about 8 h hands-on time. Along with it, an easy-to-use software package for the analysis of the derived sequencing data is provided. A-seq2 and the associated analysis software provide an efficient and reliable solution to the mapping of pre-mRNA 3&#39; ends in a wide range of conditions, from 106 or fewer cells.

This protocol describes a method for mapping pre-mRNA 3&#39; end processing sites.

Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3&#39; ...

Rapid Amplification of cDNA Ends

Source: Pablo Sanchez Bosch2, Sean Corcoran2 and Katja Br&#252;ckner1,2,3 
1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research 
2Department of Cell and Tissue Biology,&#160; 
3Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA&#160; &#160;&#160;
Rapid Amplification of cDNA Ends (RACE) is a technique that allows amplification of full-length cDNA from mRNA by extending to the 3&#8217; or 5&#8217; end, even without prior knowledge of the sequence (Frohman et al., 1988). In contrast to regular PCR, it uses only one specific PCR primer and a second non-specific primer that will indiscriminately bind to most mRNAs. Depending on whether the area to be amplified is on the 3&#8217; or 5&#8217; end of the mRNA, the second primer is chosen to bind the polyA tail (3&#8217; end) or a synthetic linker added to all transcripts (5&#8217; end). These primer combinations are known to yield &#8220;one-sided&#8221; or &#8220;anchored&#8221; PCR due to PCR amplification using the known sequence of one side- 3&#8217; or 5&#8217;(Ohara et al., 1989). The approach allows the capture of gene-specific rare mRNAs that otherwise would be hard to detect, e.g. because of their relatively low expression or unknown complete sequence (Frohman et al., 1988).
RACE is initiated with a reverse transcription step (RT) to synthetize single stranded complimentary DNA (cDNA) from mRNA. This is followed by two consecutive PCRs that amplify the cDNA fragments from a gene of interest. To perform RACE, the sequence of the gene of interest must be at least partially known, as it is needed to design the gene-specific primers (GSPs; Frohman, 1994; Liu and Gorovsky, 1993). As the second primer pair is a generic primer that will anneal to all transcripts present in the sample, the specificity of the PCR reactions is reduced. RACE is therefore ideally performed with two nested PCRs to lower the chances of amplifying non-specific transcripts (Figure 1). Depending on the direction of amplification relative to the GSP, the technique is categorized as 5&#8217; or 3&#8217; RACE (Figure 1).&#160;
3&#8217; RACE (Figure 1A) takes advantage of the poly(A) tail present in mRNAs as a generic binding site for the non-specific primer. This simplifies the technique, as one can synthetize cDNA by using oligo (dT) primers. GSPs are located in the 5&#8217; region of the transcripts. This RACE variant allows the detection of transcript variants and different 3&#8217; UTRs (Scotto Lavino et al., 2007a). In 5&#8217; RACE (Figure 1B), GSPs are located in the 3&#8217; region of the gene. To be able to use a non-specific primer that binds to all transcripts, an adapter that serves as generic primer binding site is attached to the 5&#8217; RNA ends. To attach the adapter to the mRNA, the 5&#8217; cap that protects mRNAs against exonucleases and promotes translation must be removed (Bird et al., 2016). Opposed to 3&#8217; RACE, 5&#8217; RACE helps finding differential 5&#8217; splice variants and alternative 5&#8217; UTRs (Scotto Lavino et al., 2007b).
Here we will perform 3&#8217; RACE to identify and isolate different transcript variants using the known 5&#8217; sequence (Figure 2A) encoded by the Drosophila dSmad2 (Smox) gene. dSmad2, a fly Smad protein, is an important transducer of Activin-&#946; signaling, a pathway of the TGF-&#946; superfamily. dSmad2 regulates multiple cellular processes, such as cell proliferation, apoptosis and differentiation (Upadhyay et al., 2017). As differentially spliced transcripts of dSmad2 may have different functions in the adult fly, a first step in exploring these potential functions is to assess all transcript variants of dSmad2 at this developmental stage.

Source: Pablo Sanchez Bosch2, Sean Corcoran2 and Katja Br&#252;ckner1,2,3
1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research
...

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

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