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Genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq) upon DCP1A knockout

ABSTRACT: We analyzed the effect of DCP1A knockout on nascent transcripts population in undifferentiated mouse embryonic stem cells

ORGANISM(S): Mus musculus

PROVIDER: GSE118930 | GEO | 2020/06/25

REPOSITORIES: GEO

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Genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq) upon DCP1A knockout

Project description:Genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq) upon DCP1A knockout

| PRJNA487350 | ENA

Genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq) during female mouse ES differentiation

Project description:We analyzed the effect of developmental differentiation on nascent transcripts population in female mouse embryonic stem cells.

2020-06-25 | GSE124289 | GEO

Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq).

Project description:We provide a protocol for precision nuclear run-on sequencing (PRO-seq) and its variant, PRO-cap, which map the location of active RNA polymerases (PRO-seq) or transcription start sites (TSSs) (PRO-cap) genome-wide at high resolution. The density of RNA polymerases at a particular genomic locus directly reflects the level of nascent transcription at that region. Nuclei are isolated from cells and, under nuclear run-on conditions, transcriptionally engaged RNA polymerases incorporate one or, at most, a few biotin-labeled nucleotide triphosphates (biotin-NTPs) into the 3' end of nascent RNA. The biotin-labeled nascent RNA is used to prepare sequencing libraries, which are sequenced from the 3' end to provide high-resolution positional information for the RNA polymerases. PRO-seq provides much higher sensitivity than ChIP-seq, and it generates a much larger fraction of usable sequence reads than ChIP-seq or NET-seq (native elongating transcript sequencing). Similarly to NET-seq, PRO-seq maps the RNA polymerase at up to base-pair resolution with strand specificity, but unlike NET-seq it does not require immunoprecipitation. With the protocol provided here, PRO-seq (or PRO-cap) libraries for high-throughput sequencing can be generated in 4-5 working days. The method has been applied to human, mouse, Drosophila melanogaster and Caenorhabditis elegans cells and, with slight modifications, to yeast.

| S-EPMC5502525 | biostudies-literature

Transcriptome-wide analysis upon DCP1A knockout

Project description:We analyzed the effect of DCP1A knockout on total RNA population in undifferentiated mouse embryonic stem cells

2020-06-25 | GSE118919 | GEO

Effects of Dcp1a and Dcp2 knockdown during mouse oocyte maturation

Project description:Oocyte maturation is accompanied by a transition from mRNA stability to instability. We investigated the role of DCP1A and DCP2, proteins responsible for mRNA decapping, in mRNA destabilization during mouse oocyte maturation. siRNA-mediated knockdown of both Dcp1a and Dcp2 transcripts prior to initiation of maturation inhibited the maturation-associated increase of DCP1A and DCP2, stabilized a set of maternal mRNAs that are normally degraded during maturation, and inhibited development beyond the 2-cell stage, likely a consequence of failure to activate fully the zygotic genome. Total RNA from 30 MII eggs was used in each sample. Three independent biological replicates were analyzed for each condition.

2013-02-06 | E-GEOD-27049 | biostudies-arrayexpress

PRO-seq (Precision Run-On sequencing) to profile nascent RNA of H3.3 mutant (K9A; K27A; K79A) and control mESCs

Project description:Mouse embryonic stem cells (i.e., mESCs; line ESC 129-B13) were genetically modified using CRISPR-Cas9 to mutate the H3f3b locus, in order to carry homozygous lysine-to-alanine substitution of residues K9 or K27. Two control mESC lines carrying knock-out of the H3f3a gene were used as background for the editing. To profile nascent RNA levels in H3.3 mutant and control mESCs, two rounds of PRO-seq experiment were performed. In the first round, three replicates each of H3.3K9A and control (i.e., Ctrl_9) mESCs were used. In the second round, two replicates each of H3.3K27A and control (i.e., Ctrl_27) mESCs were used. After permeabilization of the mESCs (and addition of 5% Drosophila S2 cells spike-in), a 2 biotin run-on reaction using biotin-11-UTP and biotin-11-CTP was carried out to label nascent transcripts. Biotinylated nascent RNA was then enriched as part of the library preparation process. Sequencing was performed on a NextSeq500 platform in single-end mode (75bp reads). The experiment allows for an assesment of the changes in the activity of cis-regulatory elements, upon removal of H3.3 K9 or K27 residues.

2024-09-04 | E-MTAB-12869 | biostudies-arrayexpress

Effects of Dcp1a and Dcp2 knockdown during mouse oocyte maturation

2013-02-06 | GSE27049 | GEO

DCP1A knockout: transcriptome-wide characterization

Project description:DCP1A knockout: transcriptome-wide characterization

| PRJNA487454 | ENA

Transcriptome-wide analysis upon DCP1A knockout

Project description:Transcriptome-wide analysis upon DCP1A knockout

| PRJNA487346 | ENA

Dcp1a, a novel Mek substrate, regulates the self-renewal and differentiation of mouse embryonic stem cells

Project description:Mek inhibitor is widely applied to maintain pluripotency, while prolonged Mek inhibition compromises the developmental potential of mouse embryonic stem cells (ESCs). To better understand the mechanism of Mek in pluripotency maintenance, we first demonstrated that Mek regulates gene expression at post-transcriptional steps. Consistently, many of the 66 Mek substrates identified by quantitative phosphoproteomic analysis are involved in RNA processing. We further confirmed that S563 of Dcp1a, a mRNA decapping cofactor and P-body component, is phosphorylated by Mek1. Dcp1a, as well as two other P-body components Edc4 and Dcp2, are required for the proper self-renewal and differentiation of ESCs, indicating the role of P-bodies in ESCs. Dephosphorylation of Dcp1a S563 facilitates both self-renewal and differentiation of ESCs, through promoting P-body formation and RNA storage. In summary, our study identified 66 Mek substrates supporting the Erk-independent function of Mek, and revealed that Dcp1a, phosphorylated by Mek, regulates ESC self-renewal and differentiation, through modulating P-body formation.

2024-11-19 | GSE269161 | GEO

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