Project description:To reveal the role of MCM8 in suppressing R-loop accumulation, we performed the CUT&TAG assay using the S9.6 antibody to map genome-wide R-loops in Mcm8 wildtype MEFs and Mcm8 knockout MEFs. We also conducted the CUT&TAG assay to detect genome-wide R-loops in Ddx5 downregulated MEFs by adenovirus infection and in control MEFs. To investigate the underlying molecular mechanism of MCM8 suppressing R-loops, we conducted the DNA sequencing of libraries from CUT&TAG assay using the antibody against FLAG in HEK293 cells transfected with FLAG-MCM8 plasmid and using the S9.6 antibody in HEK293 cells. Besides, an IgG control and control of RNH1 overexpression were included.
Project description:Here we describe successful implementation of CUT&Tag for profiling protein-DNA interactions in zebrafish embryos. We optimized CUT&Tag protocol to generate high resolution maps of enrichment for the histone variant H2A.Z during zebrafish development. We were able to establish dynamics of H2A.Z genomic patterning from shield stage to 24hpf embryos. Our work demonstrates the power of combining CUT&Tag with the strengths of the zebrafish system to better understand the changing embryonic chromatin landscape and its roles in shaping development.
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.