GSM7057429: ChIP_p65_KO_Control_S12; Mus musculus; ChIP-Seq
~1x107 BMDM were briefly fixed with 1% methanol-free formaldehyde solution (Thermo Scientific, # 28908) for 1 min at RT. Then, reaction was quenched with addition of 125 mM glycine for 3 min. Cells were harvested in ice-cold PBS (pH 7.4) and centrifuged at 300 x g for 5 min at 4 oC. Chromatin isolation and immunoprecipitation were carried out using CUTANATM ChIC/CUT&RUN kit (EpiCypher #141048). One hundred thousand cells were resuspended in Wash Buffer (Pre-Wash Buffer supplemented with 1X protease inhibitor cocktail, 460 mM Spermidine, 1% Triton X-100, 0.045% SDS), and incubated with activated ConA beads for 10 min at RT. Then, cells were permeabilized with 0.01% Digitonin in Wash Buffer, and incubated with 0.5 g target antibody at 4 oC overnight with agitation. The antibodies used were anti-p65 (Cell Signaling, # 8242), anti-SOCS1 (Cell Signaling, # 3950), or Normal Rabbit IgG (Cell Signaling, # 2729). Cells were washed twice with cold Wash Buffer supplemented with Digitonin, and chromatin regions around target-DNA complex were digested with pAG-MNase for 10 min at RT, followed by addition of 2 mM CaCl2, and incubation with agitation for 2 h at 4 oC. Digestion was stopped with addition of Stop Buffer, and E. coli Spike-in DNA was added to each sample for normalization. Target-DNA complex fragments were released by heat shock at 37 oC for 10 min, followed by magnetic separation of cells. Cross linking was reversed by digestion of proteins in the supernatant with 20 g Proteinase K at 55 oC overnight. Chromatin fragments were purified by standard protocol using DNA Cleanup Columns provided in the kit. IL). Sample quality, library complexity, and alignment statistics were checked using an established pipeline at the NUSeq Core Facility at Northwestern University (Chicago,