MBD2 and H3K4Me3 CUT&RUN-sequencing in C2C12 cells undergoing myogenic differentiation

MBD2 genome-binding landscape was assessed by CUT&RUN-sequencing in differentiating C2C12 cells. H3K4Me3 CUT&RUN-sequencing was performed as a positive control. Negative control experiment was also performed using a rabbit isotype control monoclonal IgG. C2C12 cells that have undergone myogenic differentiation were harvested at 0, 1, and 5 days for CUT&RUN-sequencing. C2C12 myotubes and reserve cells were separated by mild trypsinization. CUT&RUN-sequencing library was prepared using a Cell Signaling Technology CUT&RUN assay kit. Sequencing libraries were prepared using NEBNext Ultra II DNA library prep kit for Illumina and NEBNext Multiplex Oligos for Illumina (New England Biolabs, E7645 and E7600). Libraries were quantified and analyzed using Qubit dsDNA HS assay kit (Thermo Fisher Scientific) and a TapeStation 2200 with high-sensitivity DNA kit (Agilent). Libraries were sequenced in paired-end 150 base pair mode on the Illumina NovaSeq 6000 platform. Paired-end fragments were trimmed, mapped to the mm10 genome, and filtered using Trim Galore, Bowtie2 , and SAMtools , respectively. Peaks were identified using MACS2 and further filtered and analyzed with in-house Python scripts that leveraged pyBigWig and pyBedTools packages. GO term analyses (biological processes) were performed on http://metascape.org. Genomic annotation of the peaks was conducted using ChIPseeker. CUT&RUN-sequencing data were plotted using karyoploteR.