A streamlined protocol and analysis pipeline for CUT&RUN chromatin profiling
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ABSTRACT:
PROVIDER: PRJNA522731 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8581590_1.fastq.gz | Fastqsanger.gz | |||
| SRR8581590_2.fastq.gz | Fastqsanger.gz | |||
| SRR8581600_1.fastq.gz | Fastqsanger.gz | |||
| SRR8581600_2.fastq.gz | Fastqsanger.gz | |||
| SRR8581610_1.fastq.gz | Fastqsanger.gz |
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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.
2019-06-24 | GSE126612 | GEO
Project description:We generated bulk Cut&run data with antibodies agains H3K27me3 in primary mouse OPCs and OPC model cell line Oli-neu, and with antibodies agains H3K27ac in Oli-neu cell line with addition of BSA at various steps of the protocol. These datasets were used as benchmarks for the scCUT&Tag data of the mouse brain.
2021-02-10 | GSE163495 | GEO
Project description:We introduce CUT&RUNTools (https://bitbucket.org/qzhudfci/cutruntools/) as a flexible, general pipeline for facilitating the identification of chromatin-associated protein binding and genomic footprinting analysis from antibody-targeted CUT&RUN primary cleavage data. CUT&RUNTools extracts endonuclease cut site information from sequences of short read fragments and produces single-locus binding estimates, aggregate motif footprints, and informative visualizations to support the high-resolution mapping capability of CUT&RUN. We illustrate the functionality of CUT&RUNTools through analysis of CUT&RUN data acquired for GATA1, a master regulator in erythroid lineage cells. Results were compared initially to published GATA1 ChIP-seq data for cells under the same conditions. We performed de novo analysis of CUT&RUN peaks to retrieve not only GATA1’s primary motif, but also the GATA1-TAL1 composite motif, and co-factor motifs GCCCCGCCTC, CMCDCCC, and RTGASTCA that correspond to SP1, KLF1, and NFE2 co-factors. Cofactor binding was verified by independent TAL1 and KLF1 CUT&RUN, and other ChIP-seq experiments. CUT&RUNTools also generated base-pair resolution motif footprint for sequence-specific binding factors, and located likely direct binding sites by quantifying log-odds of binding scores. Overall, CUT&RUNTools should enable biologists to realize advantages of cleavage data provided by CUT&RUN, and make high-quality footprinting analysis accessible to a broad audience.
2019-08-24 | GSE136251 | GEO
Project description:Background: Identification of locus-locus contacts at the chromatin level provides a valuable foundation for understanding of nuclear architecture and function and a valuable tool for inferring long-range linkage relationships. As one approach to this, chromatin conformation capture-based techniques allow creation of genome spatial organization maps. While such approaches have been available for some time, methodological advances will be of considerable use in minimizing both time and input material required for successful application. Results: Here we report a modified tethered conformation capture protocol that utilizes a series of rapid and efficient molecular manipulations. We applied the method to Caenorhabditis elegans, obtaining chromatin interaction maps that provide a sequence-anchored delineation of salient aspects of Caenorhabditis elegans chromosome structure, demonstrating a high level of consistency in overall chromosome organization between biological samples collected under different conditions. In addition to the application of the method to defining nuclear architecture, we found the resulting chromatin interaction maps to be of sufficient resolution and sensitivity to enable detection of large-scale structural variants such as inversions or translocations. Conclusion: Our streamlined protocol provides an accelerated, robust, and broadly applicable means of generating chromatin spatial organization maps and detecting genome rearrangements without a need for cellular or chromatin fractionation. Application of modified version of TCC protocl using different C. elegans strains (N2 and glp-1) in L1, and adult life stages.
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Project description:We performed 8 C&R tests in HEK293T human cells and 8 in mouse embryonic tissues from JAX Swiss mice by using either IgG or anti-HA antibodies. To increase diversity in this test, we used both the original C&R protocol and our recently developed C&R-LoV-U version for non-DNA-binding transcriptional co-factors. The aim was to experimentally validate our generated CUT&RUN blacklists containing problematic high signal regions.
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Project description:Background: Identification of locus-locus contacts at the chromatin level provides a valuable foundation for understanding of nuclear architecture and function and a valuable tool for inferring long-range linkage relationships. As one approach to this, chromatin conformation capture-based techniques allow creation of genome spatial organization maps. While such approaches have been available for some time, methodological advances will be of considerable use in minimizing both time and input material required for successful application. Results: Here we report a modified tethered conformation capture protocol that utilizes a series of rapid and efficient molecular manipulations. We applied the method to Caenorhabditis elegans, obtaining chromatin interaction maps that provide a sequence-anchored delineation of salient aspects of Caenorhabditis elegans chromosome structure, demonstrating a high level of consistency in overall chromosome organization between biological samples collected under different conditions. In addition to the application of the method to defining nuclear architecture, we found the resulting chromatin interaction maps to be of sufficient resolution and sensitivity to enable detection of large-scale structural variants such as inversions or translocations. Conclusion: Our streamlined protocol provides an accelerated, robust, and broadly applicable means of generating chromatin spatial organization maps and detecting genome rearrangements without a need for cellular or chromatin fractionation.
2016-01-20 | GSE76930 | GEO
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.
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Project description:SOX6 CUT&RUN on HUDEP1 over expressing SOX6-Flag. The experiment is done using and anti Flag Ab to assist the genome wide binding profile of SOX6 in HUDEP1 (Human Umbilical cord blood-Derived Erythroid Progenitor-1).
2023-04-07 | E-MTAB-12800 | biostudies-arrayexpress
Project description:We examined the effects of targeting the GATA2 super-enhancer on EVI1 expression in MUTZ3. To that end, we conducted genome editing with CRISPR and assessed H3K27 acetylation with Cut&Run. The protocol described by the Henikoff group was used to generate these data.
2021-07-26 | E-MTAB-9946 | biostudies-arrayexpress