GSM7233699: H2B CUT&RUN for WT skeletal muscle stem cells rep2; Mus musculus; OTHER
In brief, FISCs were harvested and washed by cell wash buffer, then bound to concanavalin A-coated magnetic beads. Digitonin Wash Buffer was used for permeabilization. After that, cells were incubated with 5 μg of H2A or H2B antibody (Abcam) overnight at 4 °C with shaking. Then, cell-bead slurry was washed with Digitonin Wash Buffer and incubated with Protein A-MNase for 1 h at 4 °C with shaking. After washing with Digitonin Wash Buffer, CaCl2 was added into the cell-bead slurry to initiate Protein A-MNase digestion, which was then incubated in 4°C for half an hour. Then 2x Stop Buffer was added to the reaction to stop the digestion. CUT&RUN fragments were released by incubation for 30 min at 37 °C followed by centrifugation. After centrifugation, the supernatant was recovered, and the DNA purification was performed by using Phenol/Chloroform (Thermo). For DNA library construction，a NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB, E7645S) was used according to the manufacturer's instructions. Bioanalyzer analysis and qPCR were used to measure the quality of DNA libraries including the DNA size and purity. Finally, DNA libraries were sequenced on the Illumina Genome Analyzer II platform.