GSM6475269: Bap1KO_KO1_Input; Mus musculus; ChIP-Seq
Six freshly dissected mouse sciatic nerves per condition (Bap1 fl/fl/Mpz-cre and Bap1 fl/fl) were minced in 1% formaldehyde for 10min then quenched for 10min with .125M glycine. Samples were incubated in LB1 for 10min, washed with LB2, then bead-homogenized using the Next Advance Bullet Blender for 5min with speed 12 (LB1, LB2, Hung et al., 2015). Samples were centrifuged to pull down nuclei-containing pellet. Pellet was resuspended in LB3 containing 0.1% SDS (LB3, Hung et al., 2015). Samples were sonicated via Bioruptor (Diagenode) on high for 2x15min cycles (30sec on/off). Sonicated chromatin was incubated with 5ug of target antibody. A 50uL aliquot of Dynabead Protein G slurry was used to pull down the protein-antibody complex. A 10% aliquot of remaining chromatin was saved for input analysis. Immunoprecipitations were washed three times in RIPA buffer and then eluted at 65 °C in reverse cross-linking buffer (50 mM Tris, 10 mM EDTA, 1% SDS). ChIP-DNA was purified using the Cell Signaling DNA Purification Kit (CST #14209S). Antibodies used for this ChIP were Rabbit IgG (Millipore, 12-370), Rabbit H3K27me3 (Active Motif, 39155), and Rabbit H2AK119ub1 (Cell Signaling, 8240P). Libraries were prepared from ChIP DNA, and respective inputs, using the Illumina TruSeq ChIP Sample Preparation Kit.