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Optimized CUT&RUN protocol for a non-histone protein in activated primary mouse B cells

ABSTRACT: Optimized CUT&RUN protocol for a non-histone protein in activated primary mouse B cells

PROVIDER: PRJNA1130422 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR29662258_1.fastq.gz	Fastqsanger.gz
	SRR29662258_2.fastq.gz	Fastqsanger.gz
	SRR29662259_1.fastq.gz	Fastqsanger.gz
	SRR29662259_2.fastq.gz	Fastqsanger.gz
	SRR29662260_1.fastq.gz	Fastqsanger.gz

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Optimized CUT&RUN protocol for a non-histone protein in activated primary mouse B cells

Project description:ChIP-seq has long been the standard for study of chromatin-protein interactions. However, development of a new technique, CUT&RUN, shows substantial advantages compared to ChIP-seq including higher quality signal while using substantially less sample. While a powerful technique, the original protocol was unsuitable for obtaining high-quality data for non-histone proteins from activated primary B lymphocytes. To adapt this protocol for B cells, cells were fixed prior to nuclear isolation, and several adjustments were introduced to the procedure and reagents. We measured binding of RNA polymerase II to five genes and used binding of H3K4me3 histone as a positive control. Robust peaks in transcribed genes were detected with as little as 100k nuclei. Additionally, freeze-thaw of B cells prior to processing did not influence results, emphasizing the flexibility of this modified technique. Using the protocol described here will allow one to quantify low abundance proteins bound to DNA from limited numbers of B cells with more efficiency than can be achieved from the current standard, ChIP-seq.

2024-11-12 | GSE271185 | GEO

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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Project description:Over the past two decades, the dominant technology for chromatin profiling has been chromatin immunoprecipitation (ChIP), in which cellular components are solubilized to fragment chromatin, and an antibody is added to precipitate a DNA/protein complex of interest. We previously described a novel alternative to ChIP, Cleavage Under Targets & Release Using Nuclease (CUT&RUN), in which the antibody is added to permeabilized cells followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein to the antibody (PMID 29651053). Upon activation of tethered MNase, the bound complex is excised and released into the supernatant for DNA extraction and sequencing. Here we introduce four extensions to CUT&RUN: 1) a hybrid Protein A-Protein G-MNase construct that allows simplified purification using a commercial kit; 2) a modified digestion protocol that prevents release of the enzyme and so minimizes artifactual cleavage of accessible DNA; 3) a calibration strategy based on carryover of E. coli DNA introduced with the fusion protein; and 4) a novel peak-calling strategy that is customized for the low-background profiles obtained using CUT&RUN. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.

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Project description:A streamlined protocol and analysis pipeline for CUT&RUN chromatin profiling

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