Abstract
BackgroundAnalyzing the RNA pool or transcription start sites requires effective means to convert RNA into cDNA libraries for digital expression counting. With current high-speed sequencers, it is necessary to flank the cDNAs with specific adapters. Adding template-switching oligonucleotides to reverse transcription reactions is the most commonly used approach when working with very small quantities of RNA even from single cells.ResultsHere we compared the performance of DNA-RNA, DNA-LNA and DNA oligonucleotides in template-switching during nanoCAGE library preparation. Test libraries from rat muscle and HeLa cell RNA were prepared in technical triplicates and sequenced for comparison of the gene coverage and distribution of the reads within transcripts. The DNA-RNA oligonucleotide showed the highest specificity for capped 5′ ends of mRNA, whereas the DNA-LNA provided similar gene coverage with more reads falling within exons.ConclusionsWhile confirming the cap-specific preference of DNA-RNA oligonucleotides in template-switching reactions, our data indicate that DNA-LNA hybrid oligonucleotides could potentially find other applications in random RNA sequencing.
Highlights
Analyzing the RNA pool or transcription start sites requires effective means to convert RNA into cDNA libraries for digital expression counting
All oligonucleotides had a GGG-tail at the 3′ end as required for cap-dependent template-switching reactions and different barcodes followed by sequences for PCR and Illumina sequencing
The yields of the first strand cDNA synthesis reactions were measured by qPCR [2], where we found reduced cDNA yields for all reactions compared to the standard R3 template-switching oligonucleotide (Table 1)
Summary
Analyzing the RNA pool or transcription start sites requires effective means to convert RNA into cDNA libraries for digital expression counting. Adding template-switching oligonucleotides to reverse transcription reactions is the most commonly used approach when working with very small quantities of RNA even from single cells. The nanoCAGE protocol is most suitable for digital expression profiling with benchtop sequencers as it can use a lower number of reads than any RNA-seq method and avoids normalization of read counts over transcript length. NanoCAGE libraries are prepared in a two-step process comprising a reverse transcription reaction followed by stepwise PCR. During the reverse transcription reaction a template-switching oligonucleotide is added to directly introduce 5′ adapter sequences for PCR and Illumina sequencing. We prefer the use of random primers to cover non-polyadenylated transcripts in the libraries
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