CUT&RUN for Histone Modifications and Protein Targets

MM.1S cells expressing either FLAG-3xHA or FLAG-3xHA-ADA2B were subjected to light crosslinking with 0.1% formaldehyde for 1 minute, followed by quenching with 125 mM glycine. Remaining steps of CUT&RUN were performed by the Epigenomics Profiling Core at MD Anderson Cancer Center according to published protocols (Meers et al. 2019 (link)) with some modifications. Briefly, 500,000 cells were immobilized to activated Concanavalin A-coated magnetic beads (Bangs Laboratories) followed by permeabilization with wash buffer containing Digitonin (Promega). Samples were incubated with rabbit IgG (Millipore), H3K9ac (Diagenode) and HA (Cell Signaling Technology) antibodies overnight at 4°C. Targeted chromatin digestion was achieved by pAG-MNase (EpiCypher) binding for 10 minutes at room temperature followed by incubation with CaCl₂ at 4°C. DNA fragments were purified using MinElute columns (Qiagen). Libraries were prepared using NEBNext Ultra II DNA Library prep kit (New England Biolabs) following manufacturer’s instructions for H3K9ac CUT&RUN DNA, and a modified library preparation protocol for HA CUT&RUN DNA as described previously (Liu et al. 2018 (link)). Libraries were sequenced at the Advanced Technology Genomics Core at MD Anderson Cancer Center using Illumina NovaSeq 6000 SP-100 flow cell to obtain 50bp paired-end reads.