CUT&RUN for BAF155 in mus musculus: Bcl11b cKO DN23 thymocytes
Thymi from 3-6 week-old mice were removed and small cortical incisions made prior to mechanical agitation with wide-bore glass pipettes in DMEM/F-12 (Gibco) to liberate thymocytes. Bcl11b-cKO thymi were digested with 1.25 g/mL Collagenase IV (Sigma) and 1.0 g/mL DNase I (Sigma) in DMEM/F-12 for 5 min at 37C for efficient liberation of thymocytes. Cells were initially enriched via negative selection against CD8a using MACS with CD8a (Ly-2) microbeads (Miltenyi Biotec). Live, lineage-negative (DAPI-, CD11b-, CD11c-, CD19-, Gr-1-, Ter119-, NK1.1-, TCR-) DN (CD4- CD8-) cells were sorted as DN1 (CD25-, c-Kit+), DN2a (CD25+, c-Kit+),and DN3 (CD25+, c-Kit-).  CUT&RUN was performed as previously described (Skene et al., eLife, 2017) with minor modifications. Briefly, 500k cells were washed 3x in Wash Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1x EDTA-free protease inhibitor cocktail (Roche)), then bound to Concanavalin-A beads (Bangs Laboratories) according to manufacturers instructions. Cells were incubated with 1:100 dilution of anti-BAF155 antibody (G. Crabtree) overnight at 4 C in Digitonin Buffer (0.025% digitonin, 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 1x EDTA-free protease inhibitor cocktail) supplemented with 2 mM EDTA. Sample was then incubated with pA-MNase (S. Henikoff) at 1:200 dilution in Digitonin Buffer at 4 C for 1h. Digestion was performed under high Ca2+/low salt conditions in Low-Salt Rinse Buffer (3.5 mM HEPES pH 7.5, 0.5 mM spermidine, 0.025% digitonin) at 4 C for 15 min. The reaction was stopped by the addition of 4x Stop Buffer (final concentration 170 mM NaCl, 10 mM EDTA, 20 mM EGTA, 0.025% digitonin, 25 g/mL glycogen, 25 g/mL RNase A (Thermo Fisher)) and the sample was incubated at 37 C for 30 min. Sample was centrifuged at 16,000g for 10 min at 4 C, supernatant recovered, and proteins digested in 1x Stop Buffer with 0.1% SDS and 250 g /mL Proteinase K (New England Biolabs) for 1 hour at 55 C. CUT&RUN fragments were purified by phenol chloroform extraction. CUT&RUN libraries were generated using NEBNext UltraII DNA Library Prep Kit for Illumina coupled with NEBNext Multiplex Oligos for Illumina (New England Biolabs) with modifications optimized for small fragments as detailed in dx.doi.org/10.17504/protocols.io.bagaibse.