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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) during female mouse ES differentiation

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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.

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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

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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.

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A rapid, sensitive, scalable method for Precision Run-On sequencing (PRO-seq)

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