Chemical capping improves template switching and enhances sequencing of small RNAs
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ABSTRACT:
PROVIDER: PRJNA718247 | ENA |
REPOSITORIES: ENA
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Project description: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.
2021-09-13 | GSE171049 | GEO
Project description:Template-switching reverse transcription is widely used in RNA sequencing for low-input and low-quality samples, including RNA from single cells or formalin-fixed paraffin-embedded (FFPE) tissues. Previously, we identified the native eukaryotic mRNA 5' cap as a key structural element for enhancing template switching efficiency. Here, we introduce CapTS-seq, a new strategy for sequencing small RNAs that combines chemical capping and template switching. We probed a variety of non-native synthetic cap structures and found that an unmethylated guanosine triphosphate cap led to the lowest bias and highest efficiency for template switching. Through cross-examination of different nucleotides at the cap position, our data provided unequivocal evidence that the 5' cap acts as a template for the first nucleotide in reverse transcriptase-mediated post-templated addition to the emerging cDNA-a key feature to propel template switching. We deployed CapTS-seq for sequencing synthetic miRNAs, human total brain and liver FFPE RNA, and demonstrated that it consistently improves library quality for miRNAs in comparison with a gold standard template switching-based small RNA-seq kit.
| S-EPMC8754658 | biostudies-literature
Project description:RNAseq experiments of Enterovirus A71 wild type demonstrate that the pyrazine-carboxamide ribonucleotide stimulates catalyzed intra- and intermolecular template switching. These results suggest that pyrazine-2 carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination, but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.
2021-09-14 | GSE183959 | GEO
Project description:The reverse transcriptases (RTs) encoded by mobile group II intron and other non-LTR-retro-elements differ from retroviral RTs in being able to template switch from the 5' end of one template to the 3' end of another without pre-existing complementarity between the donor and acceptor nucleic acids. Here, we used the ability of a thermostable group II intron RT (TGIRT) to template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3'-DNA overhang end to establish a complete kinetic framework for the reaction and identify conditions that more efficiently capture acceptor RNAs or DNAs. The rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3'-DNA overhang complementary to the 3' nucleotide of the acceptor RNA, suggesting a role for non-templated nucleotide addition of a complementary nucleotide to the 3’ end of cDNAs synthesized from natural templates. Longer 3'-DNA overhangs progressively decrease the rate of template switching, even when complementary to the 3' end of the acceptor template. Although dependent upon only a single base pair between the donor and acceptor, template switching discriminates against mismatches, which coupled with the high processivity of the enzyme, enables the synthesis of full-length DNA copies of acceptor nucleic acids beginning directly at their 3' end. We discuss possible biological functions of the template-switching activity of group II intron and other non-LTR-retroelements RTs, as well as the optimization of this activity for adapter addition in RNA-and DNA-seq.
2019-11-11 | GSE138200 | GEO
Project description:Zhao et al. Amplification Table 1 This experiment was designed to determine the effects of template switching (TS) primer and the type of columns used in ds cDNA cleanup on the fidelity of the T7 based RNA linear amplification. BC91 total RNA was amplified with or without TS primer and with two different ds cDNA cleanup protocols. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Computed
2005-11-03 | E-GEOD-3560 | biostudies-arrayexpress
Project description:Zhao et al. Amplification Table 1 This experiment was designed to determine the effects of template switching (TS) primer and the type of columns used in ds cDNA cleanup on the fidelity of the T7 based RNA linear amplification. BC91 total RNA was amplified with or without TS primer and with two different ds cDNA cleanup protocols. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
2005-11-04 | GSE3560 | GEO
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
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
Project description:Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses
| PRJNA762534 | ENA
Project description:Capping protein controls stereocilia length and width during hair bundle development. To determine what other proteins are involved in capping protein regulation, we carried out immunoaffinity purifications targeted at either CAPZA or CAPZB2. The starting material for immunopurification was crude stereocilia membranes isolated from mouse inner ear.
2017-08-25 | PXD006077 | Pride