Here, we describe a protocol for preparing strand-specific RNA-Seq libraries that combines rRNA removal using the Ribo-Zero rRNA Removal Kit (Epicentre, Madison, WI, USA) and the dUTP method for ensuring strand specificity (Figure To synthesize the second cDNA strand, dUTP instead of deoxythymidine triphosphate (dTTP) is used, marking the second cDNA strand for subsequent degradation with uracil-DNA glycosylase (UDG) to preserve strand information In this method, the RNA is first reverse transcribed into cDNA:RNA using random primers. The deoxyuridine triphosphate (dUTP) method, one of the leading cDNA-based strategies, provides excellent library complexity, strand specificity, coverage evenness, agreement with known annotation and accuracy for expression profiling , notwithstanding the artifacts that may result from template switching or structural RNA self-priming Therefore, current strategies for transcriptome analysis all typically convert RNA to cDNA before sequencing Another strategy removes rRNA by hybridization while retaining other non-adenylated RNAs for sequencingĪlthough RNA can be sequenced directly, without conversion to cDNA, current high throughput technologies for direct RNA sequencing have short read lengths (25 to 55 nt median 33 nt) and high error rates (4%) The most widely used strategy employs poly (A) selection to enrich RNA polymerase II transcripts, but this strategy cannot be used to study RNAs lacking poly(A) tails or precursor transcripts processed into fragments that have lost their poly(A) tails for example, 7SL RNA, 7SK RNA, the 5 ′ fragment of Argonaute cleavage products, processed products of PIWI-interacting RNA (piRNA) precursors, and long non-coding RNAs such as Kcnq1ot1 in mammals Ribosomal RNAs compose an overwhelming fraction of the total RNA population (>70%) and can occupy most of the sequencing space, leaving little room for investigating other transcripts Besides measuring transcript abundance across the entire transcriptome, RNA-Seq facilitates de novo transcript annotation and assembly, quantification of splice site usage, and identification of mutations or polymorphisms between samples Strand-specific RNA sequencing (RNA-Seq) provides a powerful tool for transcriptome analysis.
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