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High efficiency and multiplexable Strand specificity RNA sequencing by Double Adapter Directional Capture

ABSTRACT: The quantification of transcriptome through RNA sequencing (RNA-seq) has widely been used to characterize all patterns of genes in a certain period. However, although many experimental protocols have recently been developed, RNA-seq is still a challenging technology in application. It is necessary to develop an easy and versatile RNA-seq method available in a wide range from single cell RNA to bulk RNA. Here we introduce a novel RNA-seq method, Double Adapter Directional Capture sequencing (DADCSeq), by directly adding adaptor at both termini of library respectively in the steps of reverse transcription and second strand synthesis, which can not only produce high quality strand specificity full-length RNA-seq library but also reduces cockamamie steps of library constructions with less time consuming. The DADCSeq was also optimized for prokaryote such as Mycobacterium smegmatis, a model bacterium for Mycobacterium tuberculosis, and can detect more genes compared to traditional methods. Taken together the DADCSeq offers a greatly simplified and cost-effective protocol with high repeatable strand specificity RNA-seq results for a diversity of RNA input materials from prokaryote to eucaryote RNA-seq libraries.

ORGANISM(S): Mus musculus Mycolicibacterium smegmatis Homo sapiens

PROVIDER: GSE237259 | GEO | 2023/07/23

REPOSITORIES: GEO

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Project description:Most eukaryotic mRNAs are enzymatically processed before they are translated, but less is known about mRNA processing in prokaryotes. While bacterial rRNAs and tRNAs are known to be highly processed, only a handful of bacterial mRNAs have been shown to be processed by ribonucleases, typically altering transcript stability. We hypothesized that mRNA processing might be more widespread in prokaryotes and constitute a previously underappreciated layer of post-transcriptional regulation. We therefore adapted RNA-seq methodologies to map transcript 5’ ends across the Mycobacterium tuberculosis transcriptome, distinguishing between transcriptional start sites (TSSs) and RNA processing sites. We report for the first time genome-wide mRNA endonucleolytic cleavage patterns in a prokaryote, and find that mRNA processing is widespread in M. tuberculosis. This has physiologic consequences, as mRNA cleavage plays a regulatory function in the ESX-1 locus by producing a transcript fragment encoding the secreted proteins EsxB and EsxA that is significantly more stable than the parent transcript, leading to greater steady-state transcript abundance. At least 20 other loci display similar patterns of processing-associated differences in transcript levels in M. tuberculosis. Mycobacteria therefore use mRNA processing to effect differential abundance of sub-sections of polycistronic transcripts, which may provide additional layers of regulation. Moreover, our results demonstrate that stable, processed mRNAs are a ubiquitous feature in this prokaryotic transcriptome, suggesting that mRNA processing may represent a major post-transcriptional regulatory mechanism. RNA from two biological replicate cultures was analyzed by RNA-seq for expression analysis and 5'-end-mapping for identification of transcriptional start sites and RNA processing sites. An additional set of three biological replicate cultures were analyzed by RNA-seq for expression analysis using a different library construction method.

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High efficiency and multiplexable Strand specificity RNA sequencing by Double Adapter Directional Capture

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