Project description:In this study, CUT&Tag-seq technology was employed to investigate MEF2A binding sites across the entire genome of chicken primary myoblasts. CUT&Tag was performed using CUT&Tag Assay Kit for Illumina Pro (TD904-1) from Vazyme. Antibody targeting MEF2A as well as IgG were used.The final DNA library on a HiSeq PE150 platform was subjected for the analyses. This study provides a wide landscape of MEF2A target genes from chicken primary myoblasts, which supports the active role of MEF2A in avian muscle development.
Project description:Identfification of MEF2A target genes using ChIP-exo in skeletla muscle and primary cardiomyocytes. Identfification of MEF2A target genes using ChIP-exo and RNA-seq in skeletal muscle and primary cardiomyocytes. MEF2 plays a profound role in the regulation of transcription in cardiac and skeletal muscle lineages. To define the overlapping and unique MEF2A genomic targets, we utilized ChIP-exo analysis of cardiomyocytes and skeletal myoblasts. Of the 2783 and 1648 MEF2A binding peaks in skeletal myoblasts and cardiomyocytes, respectively, 294 common binding sites were identified. Genomic targets were compared to differentially expressed genes in RNA-seq analysis of MEF2A depleted myogenic cells. MEF2A target genes were identified in 48 hr DM C2C12 myoblasts cells and primary cardiomyocytes using ChIP-exo. Binding profiles on MEF2A in each cell type were compared. Cross sectional-analysis between ChIP-exo identified targets and RNA-seq analysis of MEF2A deplted myoblasts was also done.
Project description:Identfification of MEF2A target genes using ChIP-exo and RNA-seq in skeletal muscle and primary cardiomyocytes. MEF2 plays a profound role in the regulation of transcription in cardiac and skeletal muscle lineages. To define the overlapping and unique MEF2A genomic targets, we utilized ChIP-exo analysis of cardiomyocytes and skeletal myoblasts. Of the 2783 and 1648 MEF2A binding peaks in skeletal myoblasts and cardiomyocytes, respectively, 294 common binding sites were identified. Genomic targets were compared to differentially expressed genes in RNA-seq analysis of MEF2A depleted myogenic cells. The effect of MEF2A gene silencing on gene expression in myoblasts was assessed at 48 hr DM. Up and downregulated genes were then compared to MEF2A target genes identified in ChIP-exo analysis of 48 hr DM C2C12 myoblasts cells and primary cardiomyocytes.
Project description:Identfification of MEF2A target genes using ChIP-exo in skeletla muscle and primary cardiomyocytes. Identfification of MEF2A target genes using ChIP-exo and RNA-seq in skeletal muscle and primary cardiomyocytes. MEF2 plays a profound role in the regulation of transcription in cardiac and skeletal muscle lineages. To define the overlapping and unique MEF2A genomic targets, we utilized ChIP-exo analysis of cardiomyocytes and skeletal myoblasts. Of the 2783 and 1648 MEF2A binding peaks in skeletal myoblasts and cardiomyocytes, respectively, 294 common binding sites were identified. Genomic targets were compared to differentially expressed genes in RNA-seq analysis of MEF2A depleted myogenic cells.
Project description:Identfification of MEF2A target genes using ChIP-exo and RNA-seq in skeletal muscle and primary cardiomyocytes. MEF2 plays a profound role in the regulation of transcription in cardiac and skeletal muscle lineages. To define the overlapping and unique MEF2A genomic targets, we utilized ChIP-exo analysis of cardiomyocytes and skeletal myoblasts. Of the 2783 and 1648 MEF2A binding peaks in skeletal myoblasts and cardiomyocytes, respectively, 294 common binding sites were identified. Genomic targets were compared to differentially expressed genes in RNA-seq analysis of MEF2A depleted myogenic cells.
Project description:Methods derived from CUT&RUN and CUT&Tag enable genome-wide mapping of the localization of proteins on chromatin from as few as one cell. These and other mapping approaches focus on one protein at a time, preventing direct measurements of co-localization of different chromatin proteins in the same cells and requiring prioritization of targets where samples are limiting. Here we describe multi-CUT&Tag, an adaptation of CUT&Tag that overcomes these hurdles by using antibody-specific barcodes to simultaneously map multiple proteins in the same cells. Highly specific multi-CUT&Tag maps of histone marks and RNA Polymerase II uncovered sites of co-localization in the same cells, active and repressed genes, and candidate cis-regulatory elements. Single-cell multi-CUT&Tag profiling facilitated identification of distinct cell types from a mixed population and inference of cell type-specific gene expression. In sum, multi-CUT&Tag increases the “per cell” information content of epigenomic maps, facilitating direct analysis of the interplay of different proteins on chromatin.
Project description:To understand the genome wide binding profile of SMAD2/3 when SMAD4 is lost, Smad2/3 ChIPseq was performed in SMAD4 knockout (S4KO) mouse embryonic stem cells (mESC) or in S4KO embryonic bodies (EBs). To figure out the effect of Dnmt3b on SMAD2/3 binding ability in the absence of SMAD4, we further performed SMAD2/3 CUT&Tag in EBs of S4KO and S4KO with Dnmt3b konckdown, as well as in mesendoderm (ME) stage of WT and S4KO. The binding sites of Dnmt3a and Dnmt3b were explored by Dnmt3a or Dnmt3b ChIP-seq and Dnmt3b CUT&Tag in WT EBs and S4KO EBs. WT with Dnmt3b knockdown and S4KO with Dnmt3b knockdown were used to as a control for the Dnmt3b CUT&Tag. Besides, we inserted a 3xFlag-tag at the C-terminus of Dnmt3a or Dnmt3b and then carried out Flag CUT&Tag in EBs.
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:We report a genome-wide mapping of DNA G-quadruplexes and histone modifications in living neurons. We used primary cultured neurons from the fetus mouse cortex. Cultured cells were treated with 2 µM Ara-C at DIV3 to prevent the proliferation of mitotic cells and were harvested at DIV10. For iG4 CUT&Tag, a BG4-expressing plasmid was transfected by electroporation at DIV0.