Abstract
BackgroundPost-transcriptional gene regulation is one of the critical layers of overall gene expression programs and microRNAs (miRNAs) play an indispensable role in this process by guiding cleavage on the messenger RNA targets. The transcriptome-wide cleavages on the target transcripts can be identified by analyzing the degradome or PARE or GMUCT libraries. However, high-throughput sequencing of PARE or degradome libraries using Illumina platform, a widely used platform, is not so straightforward. Moreover, the currently used degradome or PARE methods utilize MmeI restriction site in the 5′ RNA adapter and the resulting fragments are only 20-nt long, which often poses difficulty in distinguishing between the members of the same target gene family or distinguishing miRNA biogenesis intermediates from the primary miRNA transcripts belonging to the same miRNA family. Consequently, developing a method which can generate longer fragments from the PARE or degradome libraries which can also be sequenced easily using Illumina platform is ideal.ResultsIn this protocol, 3′ end of the 5′RNA adaptor of TruSeq small RNA library is modified by introducing EcoP15I recognition site. Correspondingly, the double-strand DNA (dsDNA) adaptor sequence is also modified to suit with the ends generated by the restriction enzyme EcoP15I. These modifications allow amplification of the degradome library by primer pairs used for small RNA library preparation, thus amenable for sequencing using Illumina platform, like small RNA library.ConclusionsDegradome library generated using this improved protocol can be sequenced easily using Illumina platform, and the resulting tag length is ~ 27-nt, which is longer than the MmeI generated fragment (20-nt) that can facilitate better accuracy in validating target transcripts belonging to the same gene family or distinguishing miRNA biogenesis intermediates of the same miRNA family. Furthermore, this improved method allows pooling and sequencing degradome libraries and small RNA libraries simultaneously using Illumina platform.
Highlights
Post-transcriptional gene regulation is one of the critical layers of overall gene expression programs and microRNAs play an indispensable role in this process by guiding cleavage on the messenger RNA targets
Using the small RNA library sequencing approach, we sequenced our degradome libraries that were of high quality (Additional file 2: Figure S2)
We further examined quality of raw reads, and 99% raw reads began with “AGCAG” (Fig. 5), which is derived from the nucleotides added to the 3′end of 5′RNA adaptor for generation of Ecop15I recognition site
Summary
Post-transcriptional gene regulation is one of the critical layers of overall gene expression programs and microRNAs (miRNAs) play an indispensable role in this process by guiding cleavage on the messenger RNA targets. This approach is time consuming, labor-intensive and costly To overcome these limitations, methods such as the PARE (parallel analysis of RNA ends) [12, 13], degradome [14] and GMUCT (genome-wide mapping of uncapped and cleaved transcripts) [15] that combine the 5′RACE and high throughput sequencing of short reads have been developed. This sequencing primer is not compatible with the standard Illumina TruSeq sequencing primer, ‘‘SR_TubeStripHyb’’ manual must be used during cluster generation [18] Another notable drawback with the currently-used degradome or PARE protocols is that these libraries yield reads or tags that are only 20-nt long, which poses difficulty in distinguishing between the members of the same target gene family
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