title: GSM8070729: KO_60m_LPS_2; Mus musculus; ChIP-Seq
library_construction_protocol: Mouse BMM and hM were crosslinked with 1% formaldehyde, scraped, PBS-washed and lysed as previously described 52. Crude nuclei were prepared, lysed 52 and sonicated for 6 cycles (hM) or 8 cycles (BMM) of 30 s on/ 30 s off in Diagenode Bioruptor Pico system. Sonicated samples are cleared with protein A/G agarose beads (Santa Cruz) and incubated with BSA-blocked protein A/G agarose beads (Santa Cruz) and antibody overnight. For control, rabbit IgG (Thermo Fisher Scientific) is used with concentration matching other antibodies. Samples were then washed 2x with Szac RIPA (150 mM NaCl, 1% v/v Nonidet P-40, 0.5% w/v Na deoxycholate, 0.1% w/v SDS, 50 mM Tris-Cl pH 8.0, 5 mM EDTA pH 8.0), 4x with Szac IP wash buffer (100 mM Tris-Cl pH 8.5, 500 mM LiCl, 1% v/v Nonidet P-40, 1% w/v Na deoxycholate), 2x with Szac RIPA buffer, and 2x with ChIP-TE buffer (100 mM Tris-Cl pH 7.5, 100 mM EDTA, 500 mM NaCl). Washed samples were next incubated with decrosslinking buffer (0.5% SDS and 0.1% v/v proteinase K in TE buffer) for 2 h at 55°C followed by overnight incubation at 65°C. DNA fragments were purified using Qiagen QIAquick PCR Purification Kit For ChIPseq, libraries were prepared with NEBNext Ultra II DNA Library Prep Kit for Illumina according to manufacturer's protocol and multiplexed with index primers (NEB E7335, E7500, E7600).