A side-by-side comparison of commercial CUT&RUN kits
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE306141 | GEO | 2026/05/15
REPOSITORIES: GEO
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Project description:Introduction: CUT&RUN (Cleavage Under Targets and Release Using Nuclease) followed by next-generation sequencing is a powerful method increasingly used in place of ChIP-seq to profile genome-wide protein-DNA interactions including those of transcription factors, histone modifications and chromatin remodelers. Although the choice of antibody is well known to affect data quality, we found that enrichment chemistry and protocol significantly affect data quality as well. Methods: In this study, we evaluated the performance of three commercially available CUT&RUN kits (Active Motif - ChIC/CUT&RUN Assay Kit, Cell Signaling Technology (CST) – CUT&RUN Assay Kit, EpiCypher - CUTANA™ ChIC/CUT&RUN Kit) on two target histone marks to represent sharp peaks (H3K4me3) and broad domains (H3K27me3). We compared data quality from libraries generated with fresh and frozen cells as well as with cell numbers that represent the low and high ends of the recommended input. Results and Discussion: Our results show that all three chemistries generate high-quality libraries. Our data revealed that despite generating libraries with fewer H3K4me3 peaks than the EpiCypher and Active Motif workflows did, the CST protocol generated libraries with superior signal and higher accuracy. Compared with the H3K4me3 libraries, the H3K27me3 libraries presented a lower percentage of shared peaks across all libraries and lower reproducibility between technical replicates. Compared with the CST protocol, the Active Motif and EpiCypher protocols generated H3K27me3 libraries with higher peak numbers, and the EpiCypher chemistry scored highest in accuracy. Libraries from cryopreserved cells generated data of similar quality as libraries from fresh cells; libraries generated with 100,000 cells had higher concordance between technical replicates than did libraries made with 500,000 cells.
2026-05-15 | GSE306233 | GEO
Project description:Purpose: Identification of glucocorticoid recepor binding sites on human kidney proximal tubular cells to better understand epigenetic alterations in diabetic kidney diseases Methods: CUT&RUN assay libraries for cultured cells or human kidneys were generated with the CUTANA kit (EpiCypher, 14-1048). 500,000 cells were mixed and incubated with Concanavalin A (ConA) conjugated paramagnetic beads. Antibodies to the glucocorticoid receptor were added to target samples and IgG was added to negative controls. The remaining steps were performed according to manufacturer’s instructions. Library preparation was performed using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs, E7645S) with manufacturer’s instructions, including minor modifications indicated by CUTANA described above. CUT&RUN libraries were sequenced on a NovaSeq instrument (Illumina, 150 bp paired-end reads). For bulk ATAC-seq, 50,000 nuclei were transposed in 25 μL of ATAC-seq transposition mix [12.5 μL 2× Illumina Tagment DNA (TD) buffer; 10.5 μL nuclease-free water; 2.0 μL Tn5 transposase (Illumina/Nextera, FC-121-1030)] and incubated at 37°C for 1 h on a thermomixer. The transposed DNA was purified with QIAGEN MinElute kit (28004) and amplified with dual indexes.
2022-07-28 | GSE195443 | GEO
Project description:Purpose: Multi-omics characterization of the consequences of deleting the SWI/SNF subunit Dpf2 in the transcriptome (RNAseq), accessibility of the genome (ATACseq) and binding of SWI/SNF subunits (CUT&RUN) in hematopoietic stem/progenitor cells, resting and polarized bone-marrow derived macrophages and resting and activated CD4+ T cells obtained from Dpf2f/f and Dpf2D/D mice. Methods: Lin- cKit+ cells were sorted from bone marrow of 28-days old Dpf2f/f and Dpf2D/D mice. Bone-marrow derived macrophages were obtained from 28-days old Dpf2f/f and Dpf2D/D mice and polarized with IFNg and LPS, or IL-4 for 24hours. CD4+ splenic T helper cells were obtained from 28-days old Dpf2f/f and Dpf2D/D mice and activated by treatment with PMA and Ionomycin. Methods: For CUT&RUN experiments, Lin- cKit+ cells were obtained from bone marrow of 28-days old Dpf2f/f and Dpf2D/D mice. Cells were crosslinked (1 min, 1% formaldehyde), quenched (125mM Glycine) and washed in buffers following the CUTANA ChIC/CUT&RUN kit according to the manufacturer’s CUT&RUN cross-linking protocol (EpiCypher, 14-1048). Results: For CUT&RUN experiments, 5 million (paired-end, 75bp) reads were obtained per sample. Pair-end fastq files were processed with the ENCODE Transcription Factor and Histone ChIP-Seq processing pipeline (https://github.com/ENCODE-DCC/chip-seq-pipeline2). Reads were trimmed using cutadapt v2.5. and aligned to the mm10 genome using Bowtie2 v2.3.4.3. SAMtools v1.9 were used to convert the output file to BAM format. Duplicates were removed using Picard Tools v2.20.7. Peak calling was performed with MACS2 v2.2.4. Conclusions: The absence of Dpf2 in LK cells results in downregulation of anti-oxidative and anti-inflammatory gene expression programs in bone marrow LK cells, bone-marrow derived macrophages and CD4+ T cells.
2023-06-06 | GSE192777 | GEO
Project description:This study uses Cleavage Under Targets and Tagmentation (Cut&Tag) chromatin profiling to map genome-wide binding sites of TES (TEAD1 Epigenetic Silencer, an engineered epigenetic silencer factor consisting of KRAB-hTEAD1(1-166)-DNMT3A/3L with V5 tag) and overexpressed wild-type TEAD1 (HA-tagged) in SNB19 glioblastoma cells. Cut&Tag was performed using the CUTANA protocol (EpiCypher). Three biological replicates were prepared for TES (detected via anti-V5) and TEAD1 (detected via anti-HA), with mouse IgG and rabbit IgG as negative controls. Peak calling identified binding sites for both factors, showing that TES largely preserves the DNA-binding specificity of wild-type TEAD1 while converting it into a transcriptional repressor.
2026-02-23 | GSE319889 | GEO
Project description:Purpose: this study is to analyze the chromatin landscape of H3R2mes in Plasmodium falciparum. Methods: In this study, H3R2me2s enrichment in the chromatin of wildtype parasite at schizont stage was analyzed by Cleavage Under Targets and Tagmentation (CUT&Tag) coupled with next generation sequencing (CUT&Tag-seq). Synchronized WT 3D7 at 40–46 hpi schizonts were harvested and fixed with 1% formaldehyde for 1 min at room temperature, followed by lysis with 0.06% saponin. The parasite pellets were suspended in 100 μl nuclear extraction buffer (20 mM HEPES–KOH pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% Glycerol, 0.5 mM Spermidine, 1x EDTA-free Protease Inhibitor cocktail). Ten μl of activated Concanavalin A-coated magnetic beads (EpiCypher # 21-1401) were added to each sample (~0.5 × 106 nuclei) and incubated for 10 min. The bead-bound nuclei were resuspended in 50 μl Antibody150 buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1× protease inhibitor cocktail, 0.01% digitonin, 2 mM EDTA) containing 0.5 μg of rabbit anti-H3R2me2s (Epigentek #A-3705-050) as the primary antibody or rabbit IgG (Cell Signaling Technology #2729S) serving as a control. The primary antibody was then removed, and nuclei were then incubated with 0.5 μg of the secondary antibodies in 50 μl of Digitonin150 buffer (Antibody150, no EDTA) at room temperature for 30 min. The secondary antibodies were mouse anti-rabbit IgG (Invitrogen #31213). The nuclei were washed thrice with 200 μl Digitonin150 Buffer for 5 min to remove unbound antibodies and finally resuspended in 50 μl Digitonin300 Buffer (Digitonin150 but with 300 mM NaCl). Then, 2.5 μl of CUTANA pAG-Tn5 (EpiCypher #15-1017) were added to each reaction at room temperature for 1 h. The nuclei were washed thrice for 5 min in 200 μl Digitonin300 Buffer to remove the unbound pA-Tn5. Next, the nuclei were resuspended in 50 μl Tagmentation buffer (10 mM MgCl2 in Digitonin300) and incubated at 37 °C for 1 h. The beads were washed with 50 µl TAPS buffer (10 mM TAPS, pH 8.5, 0.2 mM EDTA), mixed with 5 µL SDS release buffer (10 mM TAPS pH 8.5; 0.1% SDS) to quench tagmentation, and then incubated for 1 hr at 58ºC to release tagmented chromatin into solution. Finally, 15 µl of 0.67% Triton-X was added to each reaction to quench SDS. PCR with appropriate barcoded primers was performed using the CUTANA High Fidelity 2x PCR Master Mix (EpiCypher #15-1018) according to the manufacturer's recommendations. Amplified DNA libraries were captured by incubating with 1.3 × Kapa pure beads (Roche #KK8001) according to the manufacturer's recommendations, and next-generation sequencing was performed on the NextSeq 550 platform. The reads of CUT&Tag-seq from three biological replicates were mapped to the P. falciparum genome (PlasmoDB Release 39.0) by using BWA (Li and Durbin 2009) after FastQC quality control. The peak calling was finished by MACS2 version 2.2.7.1 with parameters ‘callpeak -f BAMPE’ and a q-value cutoff of 0.05. Results: The three replicates of CUT&Tag-seq using nuclei from the schizont stage showed high reproducibility, with correlation coefficients of ~0.8. Peak calling using criteria of presence in at least two of three replicates with at least 50% overlap of the peaks identified 1629 H3R2me2s signals, distributed among the 14 chromosomes. Among these peaks, 57% (927) are localized at 5’ UTR regions of 791 genes, 35% (577) at coding regions of 470 genes, and 8% (125) at 3’ UTR regions of 115 genes. The peaks are ~17.44 bp wide and ~1265 bp from the ATG sites. Comparing the transcriptome data showed genes with H3R2me2s enrichment in the 5’ UTRs showed significantly higher expression than other genes in the genome, suggesting that H3R2me2s is associated with gene activation. In addition, H3R2me2s peaks were highly co-localized with the active mark H3K9Ac, H2A.Z, and H3K4me3, further implying H3R2me2s as a euchromatin mark. Gene ontology (GO) enrichment analysis revealed a multitude of cellular pathways, including merozoite invasion, transcription, translation, stress response, cell cycle, schizogony, cell adhesion, exit from RBC, vesicle transport, signal transduction, DNA repair, and telomere organization, which are potentially regulated by this euchromatic mark. Conclusions: Collectively, H3R2me2s is a active chromatin mark and regulates invasion and other important processes in human malaria parasite.
2023-06-28 | GSE214535 | 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:We performed cleavage under targets and release using nuclease (CUT&RUN) sequencing to characterize ATAC peaks identified by snATAC-seq.
2023-04-28 | GSE213150 | GEO
Project description:Ecotropic viral integration site 1 (EVI1/MECOM) overexpression is common in myeloid malignancies. We present a new Evi1 transgenic mouse model with inducible expression in hematopoietic stem cells (HSCs) and progenitor cells (HPCs) at lower levels. Upon exogenous Evi1 induction, mice displayed anemia, thrombocytopenia, lymphopenia, and dysplasia in erythroid and megakaryocyte cells with a significant expansion of committed myeloid progenitor cells, resembling human Myelodysplastic syndrome/Myeloproliferative neoplasm (MDS/MPN)-like disease. Methods: Lin-C-Kit+ cells were isolated from 2 Evi1 overexpressing mice for CUT&RUN assay using flag antibody and IgG. Lin-C-Kit+ cells were isolated from 2 pair of WT mice and Evi1 overexpressing mice for CUT&RUN assay using H3K27me3 antibody.Around 5X105 Lin-C-Kit+ cells for each group were used in CUT&RUN assay. About 10 ng of the purified CUT&RUN DNA was used for preparation of multiplexed libraries with the NEB Ultra II DNA Library Prep Kit per manufacturer's instruction (NEB #E7103). Sequencing was conducted using an Illumina NextSeq 500 Sequencing System. Results: Molecular pathways altered in Lin-C-Kit+ cells from Evi1-OE mice. Conclusions: Evi1 has a special binding profiling in Lin-C-Kit+ cells. The modification profile of H3K27me3 was altered in Evi1-OE hematopoietic stem and progenitor cells.
2023-06-27 | GSE235439 | GEO
Project description:Cleavage Under Targets and Release Using Nuclease (CUT&RUN) has rapidly gained prominence as an effective approach for mapping protein-DNA interactions, especially histone modifications, offering substantial improvements over conventional chromatin immunoprecipitation sequencing (ChIP-seq). However, the effectiveness of this technique is contingent upon accurate peak identification, necessitating the use of optimal peak calling methods tailored to the unique characteristics of CUT&RUN data. Here, we benchmark four prominent peak calling tools - MACS2, SEACR, GoPeaks, and LanceOtron - evaluating their performance in identifying peaks from CUT&RUN datasets. Our analysis utilizes in-house data of three histone marks (H3K4me3, H3K27ac, and H3K27me3) from mouse brain tissue, as well as samples from the 4DNucleome database. We systematically assess these tools based on parameters such as the number of peaks called, peak length distribution, signal enrichment, and reproducibility across biological replicates. Our findings reveal substantial variability in peak calling efficacy, with each method demonstrating distinct strengths in sensitivity, precision, and applicability depending on the histone mark in question. These insights provide a comprehensive evaluation that will assist in selecting the most suitable peak caller for high-confidence identification of regions of interest in CUT&RUN experiments, ultimately enhancing the study of chromatin dynamics and transcriptional regulation.
2025-06-02 | GSE282809 | GEO
Project description:Sequencing of chromatin associated with H3K4me3 histone marks in live c-kit+ cells isolated by CUT&RUN from chimeras after transplantation with Ctrl-BM, MSC-BM and CpG-BM.
2023-03-24 | GSE199338 | GEO