GSM8564852: LSK_WT_Native_CUT&RUN; Mus musculus; OTHER
50,000 E17.5 HSCs pooled from 24-26 embryos or 50,000 adult bone marrow Lineage-Sca+Kit+ cells pooled from 2-3 mice were sorted into IMDM with 10% FBS. CUT&RUN protocol was followed as in Jannsens and Henikoff using protocol option 1 with modifications.15–17 Optimal digitonin concentration for primary cells was determined by titration and cell visualization with trypan blue. For fixed samples, cells were incubated with 0.1% methanol-free formaldehyde for 2 minutes and fixation was quenched with 125 mM glycine before processing. Concanavalin A magnetic beads (Bangs Laboratories #BP531) were activated with binding buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2). Cells were washed once with wash buffer (20 mM HEPES pH 7.5, 150mM NaCl, 0.5mM Spermidine, Roche Complete Protease Inhibitor EDTA-free) or cross-link wash buffer (xL wash buffer; wash buffer plus 1% Triton X-100 and 0.05% SDS) and incubated with ConA beads for 10 minutes at room temp to bind. Anti-BCLAF1 antibody (Bethyl Labs #A300-608A) was added at a 1:50 concentration in antibody buffer (wash buffer with 0.02% digitonin and 0.00008M EDTA) and incubated overnight on a nutator at 4˚C. Antibody buffer was then removed via magnet, and beads/cells were washed with dig-wash or xL dig-wash buffer (respective buffers with 0.02% digitonin). pAG-MNase (Epicypher #15-1016) was added at 1:20 concentration and incubated for 1 hour at 4˚C. Beads/cells were washed with dig-wash/xL buffer 2x and resuspended in dig-wash/xL buffer. pAG-MNase was activated by addition of 0.1 M CaCl2 and allowed to digest chromatin for 2 hours on ice. Digest was stopped by addition of 2X STOP buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% digitonin, 100 µg/ml RNAse A, 50 µg/ml glycogen). Chromatin release standard option 1 was followed. Beads/cells were incubated for 30 minutes at 37˚C and cells were collected via magnet into new tubes for DNA extraction. DNA extraction was performed via 1 hour proteinase K digest at 50˚C and phenol/chloroform extraction with phase/lock tubes (Qiagen #129056). Reverse crosslinking for fixed samples was achieved by a longer overnight proteinase K digest prior to pheno/chloroform extraction. DNA was library prepped using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645), Agencourt AMPure XP magnetic beads (Beckman Coulter #A63880), and NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1; NEB #E7600) according to manufacturer's instructions, with alterations to preserve small fragments.