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:Spatial omics emerged as a new frontier of biological and biomedical research. Here, we present spatial-CUT&Tag for spatially resolved genome-wide profiling of histone modifications by combining in situ CUT&Tag chemistry, microfluidic deterministic barcoding, and next-generation sequencing. Spatially resolved chromatin states in mouse embryos revealed tissue-type-specific epigenetic regulations in concordance with ENCODE references and provide spatial information at tissue scale. Spatial-CUT&Tag revealed epigenetic control of the cortical layer development and spatial patterning of cell types determined by histone modification in mouse brain. Single-cell epigenomes can be derived in situ by identifying 20-micrometer pixels containing only one nucleus using immunofluorescence imaging. Spatial chromatin modification profiling in tissue may offer new opportunities to study epigenetic regulation, cell function, and fate decision in normal physiology and pathogenesis.
Project description:To understand how Pou4f1 functions in RGC lineage specification and subtype formation, we performed “Cleavage Under Targets & Tagmentation” (CUT&Tag) analysis using a rabbit anti-Pou4f1 antibody and embryonic 16.5 (E16.5) retinal cells to generate barcoded PCR libraries that are enriched for Pou4f1-mediated binding. In parallel, rabbit IgG was used as a negative control for peak calling analysis, and rabbit anti-H3K9ac antibody was used to mark active enhancers and promoters.
Project description:It remains a challenge to decipher the complex relationship between DNA methylation, histone modification, and the underlying DNA sequence with limited input material. Here, we developed an efficient, low-input, and low-cost method for simultaneous profiling of genomic binding sites of histone modification and methylation status of the underlying DNA at single-base resolution from the same cells in a single experiment by integrating CUT&Tag with tagmentation-based bisulfite sequencing (CUT&Tag-BS). We demonstrated the validity of our method for both active and repressive histone modifications using 250,000 mouse ESCs. CUT&Tag-BS showed similar enrichment patterns of histone modification to those observed in non-bisulfite-treated control; it further revealed that H3K4me1-marked regions are mostly CpG-poor, lack of methylation concordance, and exhibit prevalent DNA methylation heterogeneity among the cells. We anticipate that CUT&Tag-BS will be widely applied to directly address the genomic relationship between DNA methylation and histone modification, especially in low-input scenario with precious biological samples.
Project description:This study is aimed to Investigate whether C1qbp deficiency affects the histone modification during the differentiation stage of CD8+ T cells. Transferred WT and C1qbp KO P14 cells sorted from the spleen of donor mice on day 5 post-infection with LCMV Armstrong were collected and treated for CUT-tag. CUT-tag-Seq analysis provided evidence of histone modification changes between WT and C1qbp KO CD8+ T cells. Our study shows that on day 5 post-infection with LCMV Armstrong, along with mRNA expressing profiling, the CUT-tag-seq exhibits obvious changes in H3K27me3 and H3K27Ac modification of indicated genes of WT and C1qbp KO CD8+ T cells.