GSM4654827: H3K27me3_shControl_rep1 (lane 2); Mus musculus; ChIP-Seq
GFP+ Lp30 cells were FACS-sorted fresh from murine bone marrow. Chromatin from 500,000 cells per sample was crosslinked with 1% formaldehyde (Sigma).  Samples were spiked with 25,000 HEK-293 cells, lysed and fragmented using the Diagenode Bioruptor Plus (15 cycles, 30sec on/30 sec off at high setting) to 200-700bp length following the small-cell-number protocol (PMC4328043). Chromatin  was incubated with antibodies overnight rotating at 4 degrees Celcius. The antibody-bound chromatin was captured with Protein-A sepharose beads (Sigma), washed, de-cross-linked with protease K at 65 degrees Celcius overnight as previously described (PMC4328043). Washing procedures were optimized for the individual antibodies as follows: H3K4me3-ChIP: 2x rinse and 2x wash in with RIPA-low (140 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM PMSF), followed by 2x wash in RIPA with 0.5 M NaCl (RIPA-high), 1x wash in LiCl buffer (250 mM LiCl, 10 mM Tris-HCl, 1 mM EDTA, 0.5% Tergitol, and 0.1% sodium deoxycholate),and 2x wash in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH=8). H3K4me1/H3K27ac/IgG-ChIP: 2x rinse in RIPA-low, followed by 4x wash in RIPA-low, 1x wash in LiCl buffer, and 2x wash in TE buffer. DNA libraries were prepared following NEBNext Ultra II DNA Library Kit for Illumina (New England Biolabs) guidelines and sequenced on Illumina NextSeq500.