This publication was made possible by funding from grant T32ES019851 (to C. Fritsch), R01AG054641, and an AFAR young investigator grant (to M. Vermulst).

1. Preparation
<ol>
	<li>RNases are omnipresent; therefore, clean the workspace thoroughly by spraying it down with a decontamination reagent (e.g., RNase AWAY) and 70% ethanol. Spray down any pipettes, pens, or tube racks to remove potential sources of RNase contamination.</li>
	<li>To further prevent RNases from contaminating the samples, wear a lab coat or long-sleeve T-shirt during the experimentation. Avoid contact between the gloves and the inside of any tubes that will be used, especial...

<ol>
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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+reversal+of+RNA+polymerase+II+active+site+closing+controls+fidelity+of+transcription+elongation.">Transient reversal of RNA polymerase II active site closing controls fidelity of transcription elongation.</a> Molecular Cell. 30, 557-566 (2008).</li><li>Walmacq, 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=Rpb9+subunit+controls+transcription+fidelity+by+delaying+NTP+sequestration+in+RNA+polymerase+II.">Rpb9 subunit controls transcription fidelity by delaying NTP sequestration in RNA polymerase II.</a> The Journal of Biological Chemistry. 284, 19601-19612 (2009).</li><li>Strathern, 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=The+fidelity+of+transcription:+RPB1+(RPO21)+mutations+that+increase+transcriptional+slippage+in+S.+cerevisiae.">The fidelity of transcription: RPB1 (RPO21) mutations that increase transcriptional slippage in S. cerevisiae.</a> The Journal of Biological Chemistry. 288, 2689-2699 (2013).</li><li>Irvin, J. 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=A+genetic+assay+for+transcription+errors+reveals+multilayer+control+of+RNA+polymerase+II+fidelity.">A genetic assay for transcription errors reveals multilayer control of RNA polymerase II fidelity.</a> PLoS Genetics. 10, e1004532 (2014).</li><li>Gout, J. F., 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=The+landscape+of+transcription+errors+in+eukaryotic+cells.">The landscape of transcription errors in eukaryotic cells.</a> Science Advances. 3, e1701484 (2017).</li><li>Gout, J. F., Thomas, W. K., Smith, Z., Okamoto, K., Lynch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+detection+of+in+vivo+transcription+errors.">Large-scale detection of in vivo transcription errors.</a> Proceedings of the National Academy of Science of the United States of America. 110, 18584-18589 (2013).</li><li>Viswanathan, A., You, H. J., Doetsch, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+change+caused+by+transcriptional+bypass+of+uracil+in+nondividing+cells.">Phenotypic change caused by transcriptional bypass of uracil in nondividing cells.</a> Science. 284, 159-162 (1999).</li><li>Bregeon, D., Doddridge, Z. A., You, H. J., Weiss, B., Doetsch, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+mutagenesis+induced+by+uracil+and+8-oxoguanine+in+Escherichia+coli.">Transcriptional mutagenesis induced by uracil and 8-oxoguanine in Escherichia coli.</a> Molecular Cell. 12, 959-970 (2003).</li><li>Saxowsky, T. T., Doetsch, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+polymerase+encounters+with+DNA+damage:+transcription-coupled+repair+or+transcriptional+mutagenesis.">RNA polymerase encounters with DNA damage: transcription-coupled repair or transcriptional mutagenesis.</a> Chemical Reviews. 106, 474-488 (2006).</li><li>Saxowsky, T. T., Meadows, K. L., Klungland, A., Doetsch, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=8-Oxoguanine-mediated+transcriptional+mutagenesis+causes+Ras+activation+in+mammalian+cells.">8-Oxoguanine-mediated transcriptional mutagenesis causes Ras activation in mammalian cells.</a> Proceedings of the National Academy of Science of the United States of America. 105, 18877-18882 (2008).</li><li>Vermulst, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+errors+induce+proteotoxic+stress+and+shorten+cellular+lifespan.">Transcription errors induce proteotoxic stress and shorten cellular lifespan.</a> Nature Communications. 6, 8065 (2015).</li><li>van Leeuwen, F. W., Burbach, J. P., Hol, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+in+RNA:+a+first+example+of+molecular+misreading+in+Alzheimer's+disease.">Mutations in RNA: a first example of molecular misreading in Alzheimer's disease.</a> Trends in Neurosciences. 21, 331-335 (1998).</li><li>van Leeuwen, F. W., 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=Frameshift+mutants+of+beta+amyloid+precursor+protein+and+ubiquitin-B+in+Alzheimer's+and+Down+patients.">Frameshift mutants of beta amyloid precursor protein and ubiquitin-B in Alzheimer's and Down patients.</a> Science. 279, 242-247 (1998).</li><li>Morreall, J. F., Petrova, L., Doetsch, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+mutagenesis+and+its+potential+roles+in+the+etiology+of+cancer+and+bacterial+antibiotic+resistance.">Transcriptional mutagenesis and its potential roles in the etiology of cancer and bacterial antibiotic resistance.</a> Journal of Cellular Physiology. 228, 2257-2261 (2013).</li><li>Strathern, J. N., Jin, D. J., Court, D. L., Kashlev, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+transcription+fidelity+mutants.">Isolation and characterization of transcription fidelity mutants.</a> Biochimica et Biophysica Acta. 1819, 694-699 (2012).</li><li>Acevedo, A., Andino, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Library+preparation+for+highly+accurate+population+sequencing+of+RNA+viruses.">Library preparation for highly accurate population sequencing of RNA viruses.</a> Nature Protocols. 9, 1760-1769 (2014).</li><li>Traverse, C. C., Ochman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-Wide+Spectra+of+Transcription+Insertions+and+Deletions+Reveal+That+Slippage+Depends+on+RNA:DNA+Hybrid+Complementarity.">Genome-Wide Spectra of Transcription Insertions and Deletions Reveal That Slippage Depends on RNA:DNA Hybrid Complementarity.</a> mBio. 8, (2017).</li><li>Martin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutadapt+Removes+Adapter+Sequences+From+High-Throughput+Sequencing+Reads.">Cutadapt Removes Adapter Sequences From High-Throughput Sequencing Reads.</a> EMBnet.journal. 17 (1), 10-12 (2011).</li><li>Kent, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BLAT--the+BLAST-like+alignment+tool.">BLAT--the BLAST-like alignment tool.</a> Genome Research. 12, 656-664 (2002).</li><li>Kim, 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=TopHat2:+accurate+alignment+of+transcriptomes+in+the+presence+of+insertions,+deletions+and+gene+fusions.">TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions.</a> Genome Biology. 14, R36 (2013).</li><li>Zhou, Y. 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=Isolation+and+characterization+of+RNA+polymerase+rpoB+mutations+that+alter+transcription+slippage+during+elongation+in+Escherichia+coli.">Isolation and characterization of RNA polymerase rpoB mutations that alter transcription slippage during elongation in Escherichia coli.</a> The Journal of Biological Chemistry. 288, 2700-2710 (2013).</li><li>Reid-Bayliss, K. S., Loeb, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+RNA+consensus+sequencing+for+high-fidelity+detection+of+transcriptional+mutagenesis-induced+epimutations.">Accurate RNA consensus sequencing for high-fidelity detection of transcriptional mutagenesis-induced epimutations.</a> Proceedings of the National Academy of Science of the United States of America. 114, 9415-9420 (2017).</li></ol>

The authors have nothing to disclose.

Here, we describe an optimized protocol for the preparation of C-seq libraries for the detection of transcription errors in Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol has numerous advantages over existing protocols, as well as alternative techniques.
Over the past 15 years, numerous reporter systems have been developed that rely on luciferase7,8 or Cre-Lox rec...

The genome provides a precise biological blueprint of life. To implement this blueprint correctly, it is important for the genome to be transcribed with great precision. However, transcription is unlikely to be error free. For example, RNA polymerases have long been known to be error-prone in vitro1,2, and recently it was shown that they commit errors in vivo as well3,5,6, particularly when confronted with DNA damage7,8,9,10. Taken together, these observations indicate that transcription errors occur continuously in all living cells, suggesting that they could be a potent source of mutated proteins.
This process, termed transcriptional mutagenesis, differs from classical mutagenesis in two ways. First, in contrast to genetic mutations, transcription errors affect both mitotic and post-mitotic cells, as they do not depend on DNA replication. Studying the mechanisms that impact the fidelity of transcription will, therefore, provide valuable insight into the mutation load of both mitotic and post-mitotic cells. Interestingly, transcription errors have recently been implicated in the promotion of protein aggregation11,12,13 and have been hypothesized to contribute to both carcinogenesis10 and the development of antibiotic resistance in bacteria14.
Second, in contrast to genetic mutations, transcription errors are transient in nature. Their temporary existence is particularly challenging because it makes transcription errors exceedingly difficult to detect. For example, while several labs have devised valuable reporter assays for the study of transcriptional mutagenesis, these assays are only able to measure transcription errors in a limited number of contexts and model organisms4,15. To overcome these limitations, many researchers have turned to RNA sequencing technology (RNA-seq), which theoretically allows transcription errors to be recorded throughout the transcriptome of any species. However, these studies are easily confounded by library construction artifacts, such as reverse transcription errors, PCR amplification errors, and the error-prone nature of sequencing itself. For example, reverse transcriptases commit approximately one error every ~20,000 bases, while RNA polymerases (RNAPs) are expected to make only one error every 300,000 bases5,6. Because the error rate of reverse transcription alone dwarfs the error rate of RNA polymerases inside cells, it is virtually impossible to distinguish true transcription errors from artifacts caused by the library preparation in traditional RNA-Seq data (Figure 1a).
To solve this problem, we developed an optimized version of the Circle-Sequencing (Cirseq, or C-seq henceforth) assay5,16. This assay allows the user to detect transcription errors and other rare variants in RNA throughout the transcriptome5. The circular-sequencing assay carries this name because a key step in this assay revolves around RNA circularization. Once the RNA targets are circularized, they are reverse transcribed in a rolling circle fashion, to produce linear cDNA molecules that contain numerous copies of the same RNA template. If an error was present in one of these templates, this error would also be present in every single repeat contained within the cDNA molecule. In contrast, errors introduced by reverse transcription, PCR amplification, or sequencing tend to arise randomly, and will thus be present in only one or two repeats. Thus, by generating a consensus sequence for each cDNA molecule, and distinguishing random errors from errors that occur in all repeats, library construction artifacts can effectively be separated from true transcription errors (Figure 1b).
If used properly, the C-seq assay can be used to accurately detect the rate of base substitutions, insertions, and deletions in RNA throughout the transcriptome of any species (for example, see Traverse and Ochman17). For example, we have used the C-seq assay to provide genome-wide measurements of the error rate of transcription in Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans with a single base resolution5 (unpublished observations). Originally used to accurately sequence RNA virus populations, this optimized version of the C-seq assay has been streamlined to minimize harsh conditions during the library preparation that contribute to library construction artifacts. In addition, by using a number of commercially available kits, the throughput of the assay is greatly improved, as well as its user-friendliness. If used properly, this assay can accurately detect thousands of transcription errors per replicate, thereby greatly improving on previous studies6. Overall, this method provides a powerful tool to study transcriptional mutagenesis and will allow the user to gain novel insights into the mechanisms that control the fidelity of transcription in a wide range of organisms.

<table>
	<tbody>
		<tr>
			<td>RiboPure RNA purification kit</td>
			<td>ThermoFisher</td>
			<td>AM1926</td>
			<td>Total RNA purification</td>
		</tr>
		<tr>
			<td>Genelute mRNA purification kit</td>
			<td>Sigma-Aldrich</td>
			<td>MRN70-1KT</td>
			<td>mRNA purification</td>
		</tr>
		<tr>
			<td>Nuclease-free Water</td>
			<td>Ambion</td>
			<td>AM9937</td>
			<td>Elution and dilution</td>
		</tr>
		<tr>
			<td>Ambion RNase III</td>
			<td>ThermoFisher</td>
			<td>AM2290</td>
			<td>RNA fragmentation</td>
		</tr>
		<tr>
			<td>T4 RNA Ligase 1 (ssRNA Ligase)</td>
			<td>New England Biolabs</td>
			<td>M0204S</td>
			<td>RNA circularization</td>
		</tr>
		<tr>
			<td>Ribolock</td>
			<td>ThermoFisher</td>
			<td>EO0381</td>
			<td>RNase inhibitor</td>
		</tr>
		<tr>
			<td>SuperScript III Reverse Transcriptase</td>
			<td>ThermoFisher</td>
			<td>18080044</td>
			<td>Rolling circle reverse transcription</td>
		</tr>
		<tr>
			<td>10 mM dNTP mix</td>
			<td>ThermoFisher</td>
			<td>18427013</td>
			<td>Rolling circle reverse transcription</td>
		</tr>
		<tr>
			<td>Random hexamers (50 ng/&#181;l)</td>
			<td>ThermoFisher</td>
			<td>N8080127</td>
			<td>Rolling circle reverse transcription</td>
		</tr>
		<tr>
			<td>NEB Second Strand Synthesis Module</td>
			<td>New England Biolabs</td>
			<td>E6111S</td>
			<td>Second Strand Synthesis</td>
		</tr>
		<tr>
			<td>NEBNext Ultra DNA Library Prep Kit for Illumina</td>
			<td>New England Biolabs</td>
			<td>E7370S</td>
			<td>cDNA library preparation</td>
		</tr>
		<tr>
			<td>NEB Next index primers</td>
			<td>NEB</td>
			<td>E7335S</td>
			<td>Multiplex PCR primers</td>
		</tr>
		<tr>
			<td>Oligo Clean &#38; Concentrator</td>
			<td>Zymo Research</td>
			<td>D4061</td>
			<td>Clean up of RNA and DNA samples</td>
		</tr>
		<tr>
			<td>DynaMag-2 Magnet</td>
			<td>ThermoFisher</td>
			<td>12321D</td>
			<td>Magnetic bead purification</td>
		</tr>
		<tr>
			<td>AMPure XP beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>Magnetic bead purification</td>
		</tr>
		<tr>
			<td>Eppendorf 5424 Microcentrifuge</td>
			<td>FisherScientific</td>
			<td>05-403-93</td>
			<td>centrifugation</td>
		</tr>
		<tr>
			<td>INCU-Shaker 10L</td>
			<td>Benchmark Scientific</td>
			<td>H1010</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>BIO RAD</td>
			<td>1861096</td>
			<td>Medium to High temperature cycling conditions</td>
		</tr>
		<tr>
			<td>PTC-200 Thermal Cycler</td>
			<td>GMI</td>
			<td>8252-30-0001&#160;</td>
			<td>Low temperature cycling conditions</td>
		</tr>
		<tr>
			<td>RNase Away</td>
			<td>Molecular Bioproducts</td>
			<td>700S-11</td>
			<td>Sterilization</td>
		</tr>
		<tr>
			<td>50 ml Centrifuge Tube</td>
			<td>Corning</td>
			<td>430290</td>
			<td>Nuclease-free</td>
		</tr>
		<tr>
			<td>15 ml Centrifuge Tube</td>
			<td>Corning</td>
			<td>430052</td>
			<td>Nuclease-free</td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td>Nuclease-free</td>
		</tr>
		<tr>
			<td>4200 Tapestation System</td>
			<td>Agilent</td>
			<td>G2991AA</td>
			<td>Nucleotide analysis instrument for quality control of RNA and single stranded DNA samples</td>
		</tr>
		<tr>
			<td>High Sensitivity RNA Screen Tape</td>
			<td>Agilent</td>
			<td>5067-5579</td>
			<td>Quality control of RNA and single stranded DNA samples</td>
		</tr>
		<tr>
			<td>RNA ScreenTape Sample Buffer</td>
			<td>Agilent</td>
			<td>5067-5577</td>
			<td>Quality control of RNA and single stranded DNA samples</td>
		</tr>
		<tr>
			<td>RNA ScreenTape Ladder</td>
			<td>Agilent</td>
			<td>5067-5578</td>
			<td>Quality control of RNA and single stranded DNA samples</td>
		</tr>
		<tr>
			<td>2100 Bioanalyzer Instrument</td>
			<td>Agilent</td>
			<td>G2939BA</td>
			<td>Double stranded DNA quality control</td>
		</tr>
		<tr>
			<td>High Sensitivity DNA Kit</td>
			<td>Agilent</td>
			<td>5067-4626</td>
			<td>Quality control for double stranded cDNA samples</td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>VWR</td>
			<td>462-0244</td>
			<td>Incubation</td>
		</tr>
		<tr>
			<td>NanoDrop 2000/2000C Spectrophotometer</td>
			<td>ThermoFisher</td>
			<td>ND-2000C</td>
			<td>Determination of RNA concentration</td>
		</tr>
	</tbody>
</table>

genome-wide surveillance of transcription errors in eukaryotic organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

Like all massively parallel sequencing approaches, each C-seq experiment produces an unwieldy, large dataset. For first-time users, it can be difficult to handle these datasets; thus, it is recommended that all users contact an experienced bio-informatician prior to the experimentation. On average, the expectation is that users will generate approximately 55&#8211;70 Giga bases (Gbases) per run on most massively parallel sequencing platforms. For this protocol, typically, 12&#8211;30 samp...

Watch this Scientific Journal Video about Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms at JoVE.com

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

This method can help answer key questions in the field of transcriptional mutagenesis such as which RNA polymerase subunits or biological processes control the fidelity of transcription. The main advantage of this technique is that it allows for the measurement of endogenous transcription errors in the transcriptomes of eukaryotic organisms. Generally, individuals new to this protocol will struggle because the length and complexity of the assay and a need for advanced bioinformatics in order to interpret the data.To begin the protocol, circularize the RNA fragments by heat denaturing 20 microliters of the previously prepared sample at 65 degrees Celsius for one minute. After one minute, immediately place the sample on ice for two minutes. Next, add four microliters of 10x T4 RNA ligase buffer, four microliters of 10 milliMolar ATP, one microliter of RNase inhibitor, one microliter of nuclease free water, and eight microliters of 50%polyethylene glycol, or PEG.Then mix the sample thoroughly by vortexing. Then add two microliters of 10 units per microliter of T4 RNA ligase one. Mix the sample again by pipetting and incubate it at 25 degrees Celsius for two hours in a thermocycler with the lid temperature set at 30 degrees Celsius.After the two hour incubation, add 10 microliters of nuclease free water to the sample and clean the sample with an oligo cleanup and concentration kit. Elute the sample in 20 microliters of nuclease free water. To reverse transcribe the circular RNA molecules, add four microliters of 10 milliMolar dNTPs, four microliters of 50 nanograms per microliter random hexamers, and nine microliters of nuclease free water to nine microliters of RNA.Mix thoroughly by pipetting and denature the sample at 65 degrees Celsius for one minute. After denaturing the sample, place it on ice for two minutes. Add eight microliters of 5x first strand synthesis buffer, two microliters of 0.1 milliMolar DTT, and four microliters of 200 units per microliter reverse transcriptase to the sample and mix by pipetting.Incubate the sample at 25 degrees Celsius for 10 minutes with the lid set five degrees Celsius higher than the incubation temperature. Then incubate the sample for 20 minutes at 42 degrees Celsius with the lid set at 47 degrees Celsius. Elute the sample in 42 microliters of elution solution after cleaning up with the clean up and concentrator kit.Use the second strand synthesis kit to generate a double-stranded cDNA library. Place the samples on ice and add 30 microliters of NF water to 38 microliters of your sample. Next, add eight microliters of 10x second strand buffer and four microliters of second strand enzyme and mix the sample by gentle pipetting before incubating at 16 degrees Celsius for two and a half hours.Clean up the samples with the oligo clean up and concentration kit for 100 microliter reactions and elute the samples in 38 microliters of nuclease free water. Mix the end repair reaction by pipetting and incubate it at 20 degrees Celsius for 30 minutes. Follow the 20 degree Celsius incubation with a 30 minute incubation at 65 degrees Celsius.First, dilute the adapters for next generation sequencing ten-fold in 10 milliMolar tris HCl to a final concentration of 1.5 microMolar. Then add 2.5 microliters of diluted adapter and one microliter of ligation enhancer to each sample and mix them by vortexing. Next, add 15 microliters of blunt TA ligase master mix.Pipette the sample up and down to mix and incubate it at 20 degrees Celsius for 15 minutes. Then, add three microliters of uracil specific excision reagent enzyme and incubate the sample at 37 degrees Celsius for 15 minutes. After the ligation is complete, add 13.5 microliters of nuclease free water to the sample for a total volume of 100 microliters and proceed to size selection.Acclimate magnetic beads to room temperature. Add 30 microliters of acclimated, resuspended magnetic beads to each sample and mix by pipetting. Transfer the sample to a 1.5 milliliter tube and incubate it at room temperature for five minutes.Place the sample on a magnetic stand for five minutes to separate the beads from the supernatant. Transfer the supernatant to a new 1.5 milliliter tube and dispose of the magnetic beads. Add a fresh aliquot of 15 microliters of magnetic beads to each sample.Mix thoroughly by pipetting and incubate for five minutes at room temperature. Place the samples on a magnetic rack and incubate them for five minutes at room temperature. Then carefully remove and dispose of the supernatants, making sure not to disturb the beads.Add 200 microliters of 80%freshly prepared ethanol to each of the samples without disturbing the pelleted magnetic beads and incubate for 30 seconds. Then, fully remove any trace of ethanol from the samples and air dry the beads for five minutes, but be careful not to over-dry the beads. To elute the samples from the beads, remove the tubes from the magnetic stand.Add 19 microliters of 10 milliMolar tris HCl. Pipette the mix up and down multiple times to resuspend the beads and incubate the samples for five minutes at room temperature. Place the samples back on the magnetic stand and incubate them for five minutes to separate the beads from the supernatant.Carefully remove 15 microliters of the supernatant containing the purified, size-selected cDNA libraries from the tubes and transfer it to a fresh 1.5 milliliter tube. Add five microliters of universal primer and five microliters of a unique index primer to each purified cDNA library and mix them thoroughly by pipetting. Carefully check the polymerase master mix for crystals and other precipitates and warm the master mix by hand until the precipitates dissolve.Add 25 microliters of the polymerase master mix to the sample. Mix the sample by pipetting and follow the cycling conditions listed in the accompanying text protocol for the PCR amplification. Clean up the final libraries by adding 45 microliters of resuspended magnetic beads directly to the PCR reaction.Mix them by pipetting and incubate them at room temperature for five minutes. Transfer the sample to a 1.5 milliliter tube and place it in a magnetic stand for five minutes. Following incubation, discard the supernatant and wash it twice with freshly prepared 80%ethanol for 30 seconds.After the second wash, fully remove the ethanol from the sample and air dry the bead pellet for five minutes. Then elute it in 35 microliters of 0.1x TE.Resuspend the beads by pipetting them and incubate them at room temperature for five minutes. After the tube has been on the magnetic stand for five minutes, transfer 30 microliters of the supernatant to a fresh 1.5 milliliter tube.Store the libraries at minus 80 degrees Celsius. After isolation of the RNA, two distinct rRNA peaks are visible on the electrophoretogram. Fragmentation of the RNA will result in 50 to 70 base pair RNA fragments.Rolling circle reverse transcription was used to generate cDNA molecules that are comprised of at least three tandem repeats of the circular RNA templates. Size-selection with magnetic beads allows libraries of the appropriate size to be purified. Sequencing information of a single C-seq library shows that most repeats tend to be 45 to 80 bases in length.Approximately 50%of the bases that were sequenced are part of these repeats. Since most of these bases are present in rates that contain three repeats or more, the number of unique bases that are sequenced is about one third of the total number of bases sequenced. Once it has been mastered, this technique can be done in two to three days if it is performed properly.While attempting this procedure, it is important to always maintain a sterile working environment, free of RNAses that could interfere with your work. It is also important to carefully follow each step to ensure that no mistakes are made during the procedure. After watching this video, you should have a good understanding of how to measure transcription errors in eukaryotes using the C-seq assay.

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Research

JoVE Journal

Genetics

9.3K Views.  Children's Hospital of Philadelphia. This method can help answer key questions in the field of transcriptional mutagenesis such as which RNA polymerase subunits or biological processes control the fidelity of transcription. The main advantage of this technique is that it allows for the measurement of endogenous transcription errors in the transcriptomes of eukaryotic organisms. Generally, individuals new to this protocol will struggle because the length and complexity of the assay and a need for advanced bioinformatics in order ...