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

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

PROVIDER: PRJNA632978 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11793825_1.fastq.gz	Fastqsanger.gz
	SRR11793825_2.fastq.gz	Fastqsanger.gz
	SRR11793826_1.fastq.gz	Fastqsanger.gz
	SRR11793826_2.fastq.gz	Fastqsanger.gz
	SRR11793827_1.fastq.gz	Fastqsanger.gz

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

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.

2020-05-20 | GSE150625 | GEO

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PROseq (Precision Run-On sequencing) to capture nascent RNA of wild type and mutant mouse embryonic stem cells

Project description:Precision Run-On Sequencing (PROseq) data for wild type (WT) and mutant mouse embryonic stem cells from Kreibich et al., 2022. PROseq data was obtained by permeabilizing the cells, performing a 2 biotin run-on reaction using biotin-11-UTP and biotin-11-CTP and subsequent enrichments steps for biotinylated RNA during the library preparation. This results in the enrichment of nascent transcripts, including enhancer RNA. Data in this experiment comprises PROseq data obtained from WT mouse embryonic stem cells (ES) and DNA methylation mutants (DNMT TKO ES and TET TKO ES). Comparing the PROseq data obtained from these different cell lines enables the detection of changes in the activity of cis-regulatory elements upon changes in DNA methylation.

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Project description:Efforts to examine the utility of alternate modalities for genetic results disclosure has widespread implications for how precision medicine research might yield direct health benefits for study participants. This study will examine the efficacy of an online self-guided program to return genetic results to a racial minority cohort population. Study results will provide empirical evidence on the effectiveness of alternate modalities for genetic results return, inform ongoing efforts to establish scalable approaches for effective return of genetic research results, and increase access to personal health information among African American women.

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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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A tissue dissociation method optimized for ATAC-seq and CUT&RUN in Drosophila pupal tissues [CUT&RUN]

Project description:Chromatin accessibility, histone modifications and transcription factor binding are highly dynamic during Drosophila metamorphosis and drive global changes in gene expression as larval tissues differentiate into adult structures. Unfortunately, the presence of pupa cuticle on many Drosophila tissues during metamorphosis prevents enzyme access to cells and has limited the use of enzymatic in situ methods for assessing chromatin accessibility and histone modifications. Here, we present a dissociation method for cuticle-bound pupal tissues that is optimized for use with ATAC-Seq and CUT&RUN to interrogate chromatin accessibility and histone modifications. We show this method provides comparable chromatin accessibility data to the non-enzymatic approach FAIRE-seq, with only a fraction of the amount of input tissue required. This approach is also compatible with CUT&RUN, which allows genome-wide mapping of histone modifications with less than 1/10th of the tissue input required for more conventional approaches such as Chromatin Immunoprecipitation Sequencing (ChIP-seq). Our protocol makes it possible to use newer, more sensitive enzymatic in situ approaches to interrogate gene regulatory networks during Drosophila metamorphosis.

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