Project description:TF occupancy within or near a gene promoter or enhancer region is often assumed to be sufficient evidence for the gene to be classified as a direct TF target. We identified the TF ATF3 and ONECUT2 occupancy sites and direct target candidate genes using the CUT&Tag in hepatocyte organoids.
Project description:We recently introduced CUT&Tag, an epigenomic profiling strategy in which antibodies are bound to chromatin proteins in situ in permeabilized nuclei, and then used to tether the cut-and-paste transposase Tn5. Activation of the transposase simultaneously cleaves DNA and adds DNA sequencing adapters (“tagmentation”) for paired-end DNA sequencing. Here, we introduce a streamlined CUT&Tag protocol that suppresses exposure artifacts to ensure high-fidelity mapping of the antibody-targeted protein and improves signal-to-noise over current chromatin profiling methods. Streamlined CUT&Tag can be performed in a single PCR tube from cells to amplified libraries, providing low-cost high-resolution genome-wide chromatin maps. By simplifying library preparation, CUT&Tag requires less than a day at the bench from live cells to sequencing-ready barcoded libraries. Because of low background levels, barcoded and pooled CUT&Tag libraries can be sequenced for ~$25 per sample, enabling routine genome-wide profiling of chromatin proteins and modifications that requires no special skills or equipment.
Project description:To identify the genes directly regulated by H3K9la on a genome-wide scale, we performed CUT&Tag assays using a H3K9la specific antibody in CAL27 and HN30 with or without the lactic acid treatment, and performed CUT&Tag assays using a H3K9la specific antibody in HN30 under hypoxia condition.
Project description:HIF2A(EPAS1) geenome_wide localization by Cut & Tag in MRC5 human fibroblasts ectopically expressing HIF2A in the pBabe vector, and in control MRC5 human fibroblasts containing only the pBabe vector. 2 biological replicates were done for the MRC5-HIF2A cells. Cut & Tag with an anti-H3-K27me3 Ab was done as a positive control for the Cut & Tag experiments.
Project description:We developed scNanoSeq-CUT&Tag, a streamlined method by adapting a modified CUT&Tag protocol to Oxford Nanopore sequencing platform for efficient chromatin modification profiling at single-cell resolution. We firstly tested the performance of scNanoSeq-CUT&Tag on six human cell lines: K562, 293T, GM12878, HG002, H9, HFF1 and adult mouse blood cells, it showed that scNanoSeq-CUT&Tag can accurately distinguish different cell types in vitro and in vivo. Moreover, scNanoSeq-CUT&Tag enables to effectively map the allele-specific epigenomic modifications in the human genome andallows to analyze co-occupancy of histone modifications. Taking advantage of long-read sequencing,scNanoSeq-CUT&Tag can sensitively detect epigenomic state of repetitive elements. In addition, by applying scNanoSeq-CUT&Tag to testicular cells of adult mouse B6D2F1, we demonstrated that scNanoSeq-CUT&Tag maps dynamic epigenetic state changes during mouse spermatogenesis. Finally, we exploited the epigenetic changes of human leukemia cell line K562 during DNA demethylation, it showed that NanoSeq-CUT&Tag can capture H3K27ac signals changes along DNA demethylation. Overall, we prove that scNanoSeq-CUT&Tag is a valuable tool for efficiently probing chromatin state changes within individual cells.