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

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

PROVIDER: PRJNA487350 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7741170_1.fastq.gz	Fastqsanger.gz
	SRR7741170_2.fastq.gz	Fastqsanger.gz
	SRR7741180_1.fastq.gz	Fastqsanger.gz
	SRR7741180_2.fastq.gz	Fastqsanger.gz
	SRR7741171_1.fastq.gz	Fastqsanger.gz

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

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Project description:Tracking active transcription with the nuclear run-on (NRO) assays has been instrumental in uncovering mechanisms of gene regulation. The coupling of NROs with high-throughput sequencing has facilitated the discovery of previously unannotated or undetectable RNA classes genome-wide. Precision run-on sequencing (PRO-seq) is a run-on variant that maps polymerase active sites with nucleotide or near-nucleotide resolution. One main drawback to this and many other nascent RNA detection methods is the somewhat intimidating multi-day workflow associated with creating the libraries suitable for high-throughput sequencing. Here, we present an improved PRO-seq protocol where many of the enzymatic steps are carried out while the NRO RNA remains bound to magnetic beads. These adaptations reduce time, and we demonstrate that the resulting libraries are of the same quality as libraries generated using the original published protocol. The faster library preparation procedure decreases opportunity for sample loss and RNA degradation.

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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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