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Chemical capping improves template switching and enhances sequencing of small RNAs

ABSTRACT: We deployed CapTS-seq for sequencing synthetic miRNAs, human total brain RNA, and liver FFPE RNA, and demonstrated that chemical capping in conjunction with template switching consistently reduces sequencing bias and improves library quality in comparison with commercially available RNA-seq kits. Finally, we showed the simultaneous detection of miRNAs and mRNAs in FFPE derived samples, underscoring the potential of this workflow for dissecting regulatory networks between miRNAs and their target gene transcripts.

ORGANISM(S): synthetic construct Homo sapiens

PROVIDER: GSE171049 | GEO | 2021/09/13

REPOSITORIES: GEO

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Chemical capping improves template switching and enhances sequencing of small RNAs

Project description:Chemical capping improves template switching and enhances sequencing of small RNAs

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Identification of re-capping substrates for the cytoplasmic capping enzyme complex

Project description:The analysis of capped RNAs by massively parallel sequencing has identified a large number of previously unknown transcripts, some of which are small RNAs and others are 5M-bM-^@M-^Y truncated forms of RefSeq genes. The latter may be generated by endonuclease cleavage or by stalling of Xrn1 at defined sites. With the exception of promoter-proximal transcripts the caps on all of these are added post-transcriptionally by a cytoplasmic capping enzyme complex that includes capping enzyme and a kinase that converts 5M-bM-^@M-^Y-monophosphate ends to a diphosphate capping substrate. We previously described a modified form of capping enzyme with dominant negative activity against cytoplasmic capping (DN-cCE). A tet-inducible form of this was used to identify substrates for cytoplasmic capping by treating cytoplasmic RNA from control and induced cells with and without Xrn1. Surviving RNA was analyzed on Affymetrix Human Exon 1.0 arrays and scored for changes in probe intensity as a function of its position on each RefSeq gene to derive a factor (alpha) that could be compared between sets. Notably, transcriptome-wide changes were not evident unless RNA was treated with Xrn1. This analysis identified 2,666 uncapped mRNAs in uninduced cells, 672 mRNAs that appeared in the uncapped pool in cells expressing DN-cCE, and 835 mRNAs that were in both populations. Changes in cap status of 10 re-capping targets and 5 controls were assessed by 3 independent measures; susceptibility to Xrn1, recovery with a biotin-tagged DNA primer after ligating a complementary RNA oligonucleotide to uncapped 5M-bM-^@M-^Y ends, and binding or exclusion from a high affinity cap-binding matrix comprised of immobilized eIF4E and the corresponding binding domain of eIF4G. 3 biological replicates of 4 different samples comparing XRN1 treatment/non-treatment and Dox induction/non-induction of K294A

2012-12-20 | E-GEOD-36729 | biostudies-arrayexpress

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Project description:The analysis of capped RNAs by massively parallel sequencing has identified a large number of previously unknown transcripts, some of which are small RNAs and others are 5’ truncated forms of RefSeq genes. The latter may be generated by endonuclease cleavage or by stalling of Xrn1 at defined sites. With the exception of promoter-proximal transcripts the caps on all of these are added post-transcriptionally by a cytoplasmic capping enzyme complex that includes capping enzyme and a kinase that converts 5’-monophosphate ends to a diphosphate capping substrate. We previously described a modified form of capping enzyme with dominant negative activity against cytoplasmic capping (DN-cCE). A tet-inducible form of this was used to identify substrates for cytoplasmic capping by treating cytoplasmic RNA from control and induced cells with and without Xrn1. Surviving RNA was analyzed on Affymetrix Human Exon 1.0 arrays and scored for changes in probe intensity as a function of its position on each RefSeq gene to derive a factor (alpha) that could be compared between sets. Notably, transcriptome-wide changes were not evident unless RNA was treated with Xrn1. This analysis identified 2,666 uncapped mRNAs in uninduced cells, 672 mRNAs that appeared in the uncapped pool in cells expressing DN-cCE, and 835 mRNAs that were in both populations. Changes in cap status of 10 re-capping targets and 5 controls were assessed by 3 independent measures; susceptibility to Xrn1, recovery with a biotin-tagged DNA primer after ligating a complementary RNA oligonucleotide to uncapped 5’ ends, and binding or exclusion from a high affinity cap-binding matrix comprised of immobilized eIF4E and the corresponding binding domain of eIF4G.

2012-12-20 | GSE36729 | GEO

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2019-11-11 | GSE138200 | GEO

Chemical capping improves template switching and enhances sequencing of small RNAs.

Project description: Not available

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2024-03-05 | PXD050372 | Pride

Genome-wide study of the MSL1 effect on gene expression and mRNA capping efficiency

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