Project description:We comprehensively benchmarked CUT&Tag for H3K27ac and H3K27me3 against published ChIP-seq profiles from ENCODE in K562 cells. Across a total of 30 new and 6 published CUT&Tag datasets we found that no experiment recovers more than 50% of known ENCODE peaks, regardless of the histone mark. We tested peak callers MACS2 and SEACR, identifying optimal peak calling parameters. Balancing both precision and recall of known ENCODE peaks, SEACR without retention of duplicates showed the best performance. We found that reducing PCR cycles during library preparation lowered duplication rates at the expense of ENCODE peak recovery. Despite the moderate ENCODE peak recovery, peaks identified by CUT&Tag represent the strongest ENCODE peaks and show the same functional and biological enrichments as ChIP-seq peaks identified by ENCODE. Our workflow systematically evaluates the merits of methodological adjustments and will facilitate future efforts to apply CUT&Tag in human tissues and single cells.
Project description:We comprehensively benchmarked CUT&Tag for H3K27ac and H3K27me3 against published ChIP-seq profiles from ENCODE in K562 cells. Combining multiple new and published CUT&Tag datasets, there was an average recall of 54% known ENCODE peaks for both histone modifications. To optimize data analysis steps, we tested peak callers MACS2 and SEACR and identified optimal peak calling parameters. Considering both precision and recall of known ENCODE peaks, the peak callers were comparable in their performance, although peaks produced by MACS2 match ENCODE peak width distributions more closely. We found that reducing PCR cycles during library preparation lowered duplication rates at the expense of ENCODE peak recovery. Despite the moderate ENCODE peak recovery, peaks identified by CUT&Tag represent the strongest ENCODE peaks and show the same functional and biological enrichments as ChIP-seq peaks identified by ENCODE. Our workflow systematically evaluates the merits of methodological adjustments, providing a benchmarking framework for the experimental design and analysis of CUT&Tag studies, and will facilitate future efforts to apply CUT&Tag in human tissues and single cells.
Project description:ChIP-seq. analysis of TCam-2 16 h after 10 nanomolar Romidepsin application. DMSO treated cells were used as controls. For ChIP, an antibody against histone H3 pan-acetylation was used. These data are part of the article 'The Histone Deacetylase Inhibitor Romidepsin Efficiently Targets Cisplatin-resistant Germ Cell Cancer Cells via Downregulation of the SWI/SNF-Complex Member ARID1A' (Nettersheim et al., 2016). TCam-2 cells treated for 16h with romidepsin or the solvent were fixed by formaldehyde solution and further processed by Active Motif, including DNA shearing by sonication, chromatin-immunoprecipitaion, library generation and sequencing (NextSeq 500, Illumina). Pooled input DNA of each sample including spike-in Drosophila DNA was used as controls and for normalization. The 75-nt sequence reads were mapped against the genome using BWA algorithm. Duplicate reads were removed. Only peaks that align with no more than 2 mismatches and map uniquely to the genome were used for further analysis. Intervals / peaks were identified by the MACS peak finding algorithm (cutoff p-value 1x10-7) including ENCODE blacklist filtering