This work was supported by funding from the Morgridge Institute for Research and the University of Wisconsin Foundation. We thank Krista Eastman for editorial assistance.

<ol>
	<li>Tang, F., Barbacioru, C., Wang, Y., Nordman, E., Lee, C., Xu, N., Wang, X., Bodeau, J., Tuch, B. B., Siddiqui, A., Lao, K., Surani, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA-Seq+whole-transcriptome+analysis+of+a+single+cell.">mRNA-Seq whole-transcriptome analysis of a single cell.</a> Nature Methods. 6, 377-382 (2009).</li><li>Armour, C. D., Castle, J. C., Chen, R., Babak, T., Loerch, P., Jackson, S., Shah, J. K., Dey, J., Rohl, C. A., Johnson, J. M., Raymond, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+transcriptome+profiling+using+selective+hexamer+priming+for+cDNA+synthesis.">Digital transcriptome profiling using selective hexamer priming for cDNA synthesis.</a> Nature Methods. 6, 647-649 (2009).</li><li>Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., Wold, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+and+quantifying+mammalian+transcriptomes+by+RNA-Seq.">Mapping and quantifying mammalian transcriptomes by RNA-Seq.</a> Nature Methods. 5, 621-628 (2008).</li><li>Mamanova, L., Andrews, R. M., James, K. D., Sheridan, E. M., Ellis, P. D., Langford, C. F., Ost, T. W., Collins, J. E., Turner, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRT-seq+amplification-free,+strand-specific+transcriptome+sequencing.">FRT-seq amplification-free, strand-specific transcriptome sequencing.</a> Nature Methods. 5, 130-132 (2010).</li><li>Sengupta, S., Ruotti, V., Bolin, J., Elwell, A., Hernandez, A., Thomson, J., Stewart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+consistent,+fully+representative+mRNA-Seq+libraries+from+ten+nanograms+of+total+RNA.">Highly consistent, fully representative mRNA-Seq libraries from ten nanograms of total RNA.</a> Biotechniques. 49, 898-904 (2010).</li><li>Langmead, B., Trapnell, C., Pop, M., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+and+memory-efficient+alignment+of+short+DNA+sequences+to+the+human+genome.">Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.</a> Genome. Biology. 10, R25-R25 (2009).</li><li>Li, B., Ruotti, V., Stewart, R. M., Thomson, J. A., Dewey, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq+gene+expression+estimation+with+read+mapping+uncertainty.">RNA-Seq gene expression estimation with read mapping uncertainty.</a> Biotechniques. 26, 493-500 (2010).</li><li>Thomson, J. A., Itskovitz-Eldor, J., Shapiro, S. S., Waknitz, M. A., Swiergiel, J. J., Marshall, V. S., Jones, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li></ol>

The authors disclose that J.A.T. is a founder, stockowner, consultant and board member of Cellular Dynamics International (CDI). He also serves as scientific advisor to and has financial interests in Tactics II Stem Cell Ventures.

Current protocols for making paired end libraries require between 1 &mu;g9 to 2.5 &mu;g10 starting amount of total RNA. Here we present our linear T7 amplification based (T7LA) method to prepare both single read and paired end Illumina sequencing libraries and show that this method allows generation of libraries from as low as 10 ng of total RNA, producing data that is comparable to that of minimally amplified (MinAmp) libraries made from 1000 fold more starting material (10 &mu;g total RNA). The 10 ng libraries not only identify similar total numbers of genes, but also produce gene expression signatures that are similar (Figures 3a and b). Moreover, both the single read and paired end libraries produced by the T7LA method are very similar to each other (Figure 3c), which allows researchers to compare data generated by libraries made from either protocol. Since these libraries were prepared from human embryonic stem cell RNA, we searched for 30 stem cell specific genes among the libraries and find that almost all of these genes (93-100%) are identified by the libraries made from at least 10 ng of starting total RNA (Figure 4), thus validating our protocol. We believe our protocol would be very useful for researchers, especially in circumstances such as flow sorted cells or laser-micro-dissected tissue where starting material is limiting. In such circumstances, our protocol would allow generation of gene expression data comparable to libraries made from much larger starting quantities since our protocol produces expression profiles comparable across at least 3 orders of magnitude of starting RNA.

<table border="1">
<tbody>
<tr>
<td>Company	</td>
<td>Kit/Reagent</td>
<td>Catalog #</td>
<td>Special Comments</td>
</tr>
<tr>
<td>Ambion	</td>
<td>Fragmentation reagent</td>
<td>AM8740</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ambion	</td>
<td>Liner Acrylamide</td>
<td>AM9520</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ambion</td>
<td>MEGAscript T7 Kit</td>
<td>AM1334</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Ambion</td>
<td>Non DEPC treated nuclease free water</td>
<td>AM9932</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fermentas</td>
<td>dNTP set</td>
<td>R0181</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Fermentas</td>
<td>methylated dNTPs</td>
<td>R0431</td>
<td>For Single Read sample prep only</td>
</tr>
<tr>
<td>Illumina</td>
<td>TruSeq SR Cluster Generation kit v5</td>
<td>GD-203-5001</td>
<td>For Single Read sample prep only</td>
</tr>
<tr>
<td>Illumina		</td>
<td>TruSeq Seq Kit v5 36 cycles</td>
<td>FC-104-5001</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Illumina</td>
<td>Chip Seq Sample Prep Kit</td>
<td>IP-102-1001</td>
<td>For Single Read sample prep only. Can be replaced with NEB DNA sample prep Master Mix Set 1 Cat# E6040S</td>
</tr>
<tr>
<td>Illumina			</td>
<td>TruSeq PE Cluster Generation kit v5</td>
<td>PE-203-5001</td>
<td>For paired end sample prep only</td>
</tr>
<tr>
<td>Illumina			</td>
<td>Paired End Sample Prep Kit</td>
<td>PE-102-1001</td>
<td>For paired end sample prep only</td>
</tr>
<tr>
<td>Invitrogen		</td>
<td>1kb plus ladder</td>
<td>10787-018</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Invitrogen		</td>
<td>E. coli Rnase H</td>
<td>18021-014</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Invitrogen		</td>
<td>Rnase Out</td>
<td>10777-019</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Invitrogen		</td>
<td>2nd strand buffer 5x</td>
<td>10812-014</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Invitrogen		</td>
<td>E. coli DNA polymerase</td>
<td>18010-017</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Invitrogen		</td>
<td>E-gel SYBR safe 2%</td>
<td>G521802</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Invitrogen		</td>
<td>Superscript III (with 5X FS buffer and 0.1M DTT)</td>
<td>18080-085</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Invitrogen		</td>
<td>Trizol</td>
<td>12183555</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Invitrogen		</td>
<td>RNA quibit assay kit</td>
<td>Q32852</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Invitrogen		</td>
<td>dsDNA HS qubit assay kit</td>
<td>Q32851</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Invitrogen		</td>
<td>E. coli DNA ligase</td>
<td>18052-019</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Invitrogen		</td>
<td>T4 DNA Polymerase</td>
<td>18005-025</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Invitrogen		</td>
<td>Ultra Pure Dnase Rnase water</td>
<td>10977-015</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Invitrogen		</td>
<td>Superscript II double stranded cDNA synthesis kit</td>
<td>11917-020</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Invitrogen			</td>
<td>Random Primer (invitrogen)</td>
<td>48190-011</td>
<td>For paired end sample prep only</td>
</tr>
<tr>
<td>IDT			</td>
<td>Not1Nonamer B primer</td>
<td>N/A</td>
<td>For Single Read sample prep only</td>
</tr>
<tr>
<td>IDT		</td>
<td>Oligo dT T7</td>
<td>N/A</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>NEB			</td>
<td>Not1 digestion kit</td>
<td>R0189S</td>
<td>For Single Read sample prep only</td>
</tr>
<tr>
<td>Qiagen		</td>
<td>Rneasy Minelute kit</td>
<td>74204</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Qiagen		</td>
<td>RNEasy Mini Kit (50)</td>
<td>74104</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Qiagen		</td>
<td>Gel purification kit</td>
<td>28604</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Qiagen		</td>
<td>Dnase set</td>
<td>79254</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Qiagen		</td>
<td>Rneasy MinElute kit</td>
<td>74204</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Zymo Research		</td>
<td>DNA clean and concentrator (250X)</td>
<td>D4014</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>

single read and paired end mrna-seq illumina libraries from 10 nanograms total rna

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Watch this Scientific Journal Video about Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA at JoVE.com

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

single-read-paired-end-mrna-seq-illumina-libraries-from-10-nanograms

Research

JoVE Journal

Biology

39.0K Views.  Morgridge Institute for Research. Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.