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, 3:9

Non-coding RNARNA: Methods and Protocols

Abstract

BackgroundHigh throughput DNA sequencing technology has enabled quantification of all the RNAs in a cell or tissue, a method widely known as RNA sequencing RNA-Seq. However, non-coding RNAs such as rRNA are highly abundant and can consume >70% of sequencing reads. A common approach is to extract only polyadenylated mRNA; however, such approaches are blind to RNAs with short or no polyA tails, leading to an incomplete view of the transcriptome. Another challenge of preparing RNA-Seq libraries is to preserve the strand information of the RNAs.

DesignHere, we describe a procedure for preparing RNA-Seq libraries from 1 to 4 μg total RNA without polyA selection. Our method combines the deoxyuridine triphosphate dUTP-uracil-DNA glycosylase UDG strategy to achieve strand specificity with AMPure XP magnetic beads to perform size selection. Together, these steps eliminate gel purification, allowing a library to be made in less than two days. We barcode each library during the final PCR amplification step, allowing several samples to be sequenced in a single lane without sacrificing read length. Libraries prepared using this protocol are compatible with Illumina GAII, GAIIx and HiSeq 2000 platforms.

DiscussionThe RNA-Seq protocol described here yields strand-specific transcriptome libraries without polyA selection, which provide approximately 90% mappable sequences. Typically, more than 85% of mapped reads correspond to protein-coding genes and only 6% derive from non-coding RNAs. The protocol has been used to measure RNA transcript identity and abundance in tissues from flies, mice, rats, chickens, and frogs, demonstrating its general applicability.

KeywordsRNA-Seq Transcriptome High throughput sequencing AbbreviationsdATPDeoxyadenosine triphosphate

dCTPDeoxycytidine triphosphate

dGTPDeoxyguanosine triphosphate

DTTDL-dithiothreitol

dTTPDeoxythymidine triphosphate

dUTPDeoxyuridine triphosphate

LBLuria-Bertani

NEBNew England Biolabs

PCRPolymerase chain reaction

PEGPolyethylene glycol

piRNAPiwi-interacting RNA

RNA-SeqRNA sequencing

RNase HRibonuclease H

rRNARibosomal RNA

snoRNASmall nucleolar RNA

snRNASmall nuclear RNA

SOCSuper optimal broth with catabolite repression

T4 PNKT4 Polynucleotide Kinase

UDGUracil-DNA glycosylase.

Electronic supplementary materialThe online version of this article doi:10.1186-1758-907X-3-9 contains supplementary material, which is available to authorized users.

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Author: Zhao Zhang - William E Theurkauf - Zhiping Weng - Phillip D Zamore

Source: https://link.springer.com/article/10.1186/1758-907X-3-9







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