Dataset Information

Comparison of Poly-A+ Selection and rRNA Depletion in Detection of lncRNA in Two Equine Tissues Using RNA-seq.

ABSTRACT: Long non-coding RNAs (lncRNAs) are untranslated regulatory transcripts longer than 200 nucleotides that can play a role in transcriptional, post-translational, and epigenetic regulation. Traditionally, RNA-sequencing (RNA-seq) libraries have been created by isolating transcriptomic RNA via poly-A+ selection. In the past 10 years, methods to perform ribosomal RNA (rRNA) depletion of total RNA have been developed as an alternative, aiming for better coverage of whole transcriptomic RNA, both polyadenylated and non-polyadenylated transcripts. The purpose of this study was to determine which library preparation method is optimal for lncRNA investigations in the horse. Using liver and cerebral parietal lobe tissues from two healthy Thoroughbred mares, RNA-seq libraries were prepared using standard poly-A+ selection and rRNA-depletion methods. Averaging the two biologic replicates, poly-A+ selection yielded 327 and 773 more unique lncRNA transcripts for liver and parietal lobe, respectively. More lncRNA were found to be unique to poly-A+ selected libraries, and rRNA-depletion identified small nucleolar RNA (snoRNA) to have a higher relative expression than in the poly-A+ selected libraries. Overall, poly-A+ selection provides a more thorough identification of total lncRNA in equine tissues while rRNA-depletion may allow for easier detection of snoRNAs.

SUBMITTER: Dahlgren AR

PROVIDER: S-EPMC7549351 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Publications

Comparison of Poly-A<sup>+</sup> Selection and rRNA Depletion in Detection of lncRNA in Two Equine Tissues Using RNA-seq.

Dahlgren Anna R AR Scott Erica Y EY Mansour Tamer T Hales Erin N EN Ross Pablo J PJ Kalbfleisch Theodore S TS MacLeod James N JN Petersen Jessica L JL Bellone Rebecca R RR Finno Carrie J CJ

Non-coding RNA 20200821 3

Long non-coding RNAs (lncRNAs) are untranslated regulatory transcripts longer than 200 nucleotides that can play a role in transcriptional, post-translational, and epigenetic regulation. Traditionally, RNA-sequencing (RNA-seq) libraries have been created by isolating transcriptomic RNA via poly-A<sup>+</sup> selection. In the past 10 years, methods to perform ribosomal RNA (rRNA) depletion of total RNA have been developed as an alternative, aiming for better coverage of whole transcriptomic RNA, ...[more]

PMID: 32825772

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Project description:RNA sequencing (RNA-Seq) is often used for transcriptome profiling as well as the identification of novel transcripts and alternative splicing events. Typically, RNA-Seq libraries are prepared from total RNA using poly(A) enrichment of the mRNA (mRNA-Seq) to remove ribosomal RNA (rRNA), however, this method fails to capture non-poly(A) transcripts or partially degraded mRNAs. Hence, a mRNA-Seq protocol will not be compatible for use with RNAs coming from Formalin-Fixed and Paraffin-Embedded (FFPE) samples.To address the desire to perform RNA-Seq on FFPE materials, we evaluated two different library preparation protocols that could be compatible for use with small RNA fragments. We obtained paired Fresh Frozen (FF) and FFPE RNAs from multiple tumors and subjected these to different gene expression profiling methods. We tested 11 human breast tumor samples using: (a) FF RNAs by microarray, mRNA-Seq, Ribo-Zero-Seq and DSN-Seq (Duplex-Specific Nuclease) and (b) FFPE RNAs by Ribo-Zero-Seq and DSN-Seq. We also performed these different RNA-Seq protocols using 10 TCGA tumors as a validation set.The data from paired RNA samples showed high concordance in transcript quantification across all protocols and between FF and FFPE RNAs. In both FF and FFPE, Ribo-Zero-Seq removed rRNA with comparable efficiency as mRNA-Seq, and it provided an equivalent or less biased coverage on gene 3' ends. Compared to mRNA-Seq where 69% of bases were mapped to the transcriptome, DSN-Seq and Ribo-Zero-Seq contained significantly fewer reads mapping to the transcriptome (20-30%); in these RNA-Seq protocols, many if not most reads mapped to intronic regions. Approximately 14 million reads in mRNA-Seq and 45-65 million reads in Ribo-Zero-Seq or DSN-Seq were required to achieve the same gene detection levels as a standard Agilent DNA microarray.Our results demonstrate that compared to mRNA-Seq and microarrays, Ribo-Zero-Seq provides equivalent rRNA removal efficiency, coverage uniformity, genome-based mapped reads, and consistently high quality quantification of transcripts. Moreover, Ribo-Zero-Seq and DSN-Seq have consistent transcript quantification using FFPE RNAs, suggesting that RNA-Seq can be used with FFPE-derived RNAs for gene expression profiling.

Dataset Information

Comparison of Poly-A+ Selection and rRNA Depletion in Detection of lncRNA in Two Equine Tissues Using RNA-seq.

Publications

Comparison of Poly-A<sup>+</sup> Selection and rRNA Depletion in Detection of lncRNA in Two Equine Tissues Using RNA-seq.

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