P3-CM CUT&RUN-Tip60
CUT&RUN was performed on primary neonatal CMs using the CUT&RUN assay kit (Cell Signaling Technology, Cat. No. 86652) according to the manufacturers instructions. Briefly, 110^5 live primary neonatal CMs were harvested, washed, and bound to concanavalin A-coated magnetic beads to immobilize the cells. Bead-bound cells were then permeabilized and incubated with a primary antibody specific to the target protein at 4C overnight with gentle agitation. After washing to remove unbound antibody, cells were incubated with a recombinant pAG-MNase enzyme for 1 hour at 4C to tether the nuclease to the antibody-bound chromatin. Targeted chromatin cleavage was initiated by the addition of calcium chloride and carried out at 4C for 30 minutes. The reaction was stopped by adding stop buffer, and the released chromatin fragments were collected from the supernatant. The input control sample (antibody untreated) was processed in parallel to assess background cleavage and to normalize signal enrichment. DNA was purified using spin columns provided in the kit and sequencing libraries were prepared according to the instructions of the DNA Library Prep Kit for Illumina (New England Biolabs, Cat. No. E7645). Sequencing was performed in a paired-end 150 bp configuration on the Illumina NovaSeq system, following manufacturers instructions.