mRNA sequencing Procedure from nanogram quantity of total RNA
Prior to initiating sequencing procedure, the quality of total RNA was analyzed on Agilent Bioanalyzer.
The RNA sample that has more than 7.0 RNA integration number (RIN) was taken up for seq library
preparation using mRNA Seq Kit supplied by Illumina (Cat # 1004814). The protocol followed was as per
manufacturer’s instruction with following modifications for mRNA isolation from nanograms levels of
total RNA as starting material. Strict measures were taken to create RNase free working conditions to
prepare sequencing libraries from as low as 5-20 nanograms of total RNA as starting material. Total
working area, pipette aids and required equipment were thoroughly wiped with RNase Away, and all the
tips and eppendorf tubes used were RNase free. Briefly, mRNA was isolated from total RNA using 7
microliters of oligo dT on Sera-magnetic beads and 50 microliters of binding buffer. The mRNA was then
fragmented in the presence of divalent cations at 940C. The fragmented RNA was converted into double
stranded cDNA. After polishing the ends of the cDNA, adenine base was added at the 3’ ends following
which Illumina supplied specific adaptors were ligated. The adaptor ligated DNA was amplified by 15
cycle PCR. The PCR DNA was then purified on Qiagen PCR purification kit to get the final seq library
ready for sequencing. The insert size and DNA concentration of the seq library was determined on
Agilent Bioanalyzer. Each RNA seq library was layered on one of the eight lanes of the Illumina flow cell
at appropriate concentration and bridge amplified to get around 40- 45 million raw reads. The DNA
reads on the flow cell were then sequenced on Genome Analyzer IIx using appropriate base pair
sequencing recipe. At the current rate, on an average 33-37 million passing filter reads per lane were
obtained on the genome Analyzer IIx, which yield 2.3 to 3.7 billion sequenced bases using 100 bp single
end sequencing recipe. The rate of sequencing was around 1.2 hours per base. Basically, sequencing by
synthesis procedure was followed by incorporation of one fluorescent labeled base at a time. At the
addition of each base, the fluorescence images were captured by the in situ camera. The images were
converted into intensities that in turn were called for bases. The entire process of imaging to base calling
was carried out real time on the computer attached to the genome analyzer. The data was then
transferred and stored in Yale HPC server Bulldog-N. The quality of the sequence and its alignment to
reference genome was carried out by the Illumina supported Consensus Assessment of Sequence and
Variation (CASAVA) software program.
Sequencing by RNA Amplification:
YCGA has expertise in sequencing the mRNA from as little as 1-2 nanograms of total
RNA. Prior to seq library sample preparation, total RNA is amplified using Ovation RNA-Seq
System (NuGen Cat. # 7100-08) that preserves the transcriptome profile even after
amplification. The Ovation RNA-Seq System provides a fast and simple method for preparing
microgram quantities of double stranded cDNA from as little as 1.0 nanogram of total RNA that
can be integrated with Illumina’s RNA-Seq library preparation protocol from end repair step.
Amplification is initiated at the 3' end as well as randomly throughout the whole transcriptome
to evenly cover 5’ and 3’ portions of the transcripts. The Ovation RNA-Seq System uses RiboSPIA technology involving proprietary combination of reverse transcriptase and primers for
preferential priming and subsequent amplification of non-rRNA sequences. Reads mapping to
rRNA sequences are minor, which fall in the range of 3.0% to 4%.
Download

Experimental Procedures for Grant Write-Up (Illumina