Project description:Targeted epigenomic profiling methods CUT&RUN and CUT&Tag were used to examine TASOR genome binding and TASOR-regulated H3K9me3 deposition on chromatin.
Project description:We generated bulk Cut&run data with antibodies agains H3K27me3 in primary mouse OPCs and OPC model cell line Oli-neu, and with antibodies agains H3K27ac in Oli-neu cell line with addition of BSA at various steps of the protocol. These datasets were used as benchmarks for the scCUT&Tag data of the mouse brain.
Project description:To understand the functional role of PRC1 in defining the epigenomic and transcriptional landscape in Aundiff, we eliminated PRC1 function in germ cells using Ddx4-Cre to mutate the E3 ubiquitin ligase RNF2 on a background lacking its partially redundant paralog RING1 (RING1A) to create PRC1 conditional knockout (PRC1cKO) mice. We discover a key role of Polycomb complexes in shielding the undifferentiated state of mouse adult spermatogonia. CUT&RUN or CUT&Tag for H2AK119ub, H3K27me3 and H3K4me3 in WT, PRC1ctrl and PRC1cKO spermatogonial cells.
Project description:Here we describe successful implementation of CUT&RUN for profiling protein-DNA interactions in zebrafish embryos. We apply modified a CUT&RUN method to generate high resolution maps of enrichment for H3K4me3, H3K27me3, H3K9me3, and RNA polymerase II during zebrafish gastrulation. Using this data, we identify a conserved subset of developmental genes that are enriched in both H3K4me3 and H3K27me3 during gastrulation, and we demonstrate the increased effectiveness of CUT&RUN for detecting protein enrichment at repetitive sequences with reduced mappability. Our work demonstrates the power of combining CUT&RUN with the strengths of the zebrafish system to better understand the changing embryonic chromatin landscape and its roles in shaping development.
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 have established a pluripotent stem cell (PSC)-based 3D model of human axial development, which recapitulates in vitro key features of the somitogenesis process including axial elongation, segmentation and sequential formation of epithelial somites with proper rostrocaudal pattering, and traveling wave-like gene expression dynamics associated with the segmentation clock. Initial molecular and functional analysis of these axially-organized embryonic tail-like structures, which we termed Axioloids, revealed the spatiotemporally controlled self-organization of somitogenesis associated human mesodermal cell populations. In order to gain a deeper understanding of this model, we performed CUT&Tag experiments using anti-H3K4me3 and anti-H3K27me3 antibodies to analyze the epigenomic landscapes in the Axioloids.
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.