title: GSM2974758: NOD_BMDM_H3K27ac_KLA_1h_rep1; Mus musculus; ChIP-Seq
library_construction_protocol: Femur, tibia and iliac bones from the different mouse strains were flushed with DMEM high glucose (Corning) and red blood cells were lysed using red blood cell lysis buffer (eBioscience). After counting, 20 million bone marrow cells were seeded per 15cm non-tissue culture plates in DMEM high glucose (50%) with 20% FBS (Omega Biosciences), 30% L929-cell conditioned media (as source of M-CSF), 100 U/ml penicillin/streptomycin+L-glutamine (Gibco) and 2.5μg/ml Amphotericin B (HyClone). After 4 days of differentiation, 16.7 ng/ml mouse M-CSF (Shenandoah Biotechnology) was added to the media. After an additional 2 days of culture, non-adherent cells were washed off with room temperature DMEM high glucose and macrophages were obtained as a homogeneous population of adherent cells which were scraped and subsequently seeded onto tissue culture-treated petri dishes overnight in DMEM high glucose containing 10% FBS, 100 U/ml penicillin/streptomycin+L-glutamine, 2.5μg/ml Amphotericin B and 16.7 ng/ml M-CSF. For KLA activation, macrophages were treated with 10 ng/mL KLA (Avanti Polar Lipids) for 1 or 6 hours. ChIP-Seq: Chromatin immunoprecipitation (ChIP) was performed as described previously (Oishi et al., 2017). In brief, cells were resuspended in swelling buffer (10mM HEPES/KOH (pH7.9), 85mM KCl, 1mM EDTA, 0.5% IGEPAL CA-630) with protease inhibitors for 5min and then spun down and resuspended in 500μl lysis buffer (50mM Tris/HCl (pH7.4), 1% SDS, 0.5% Empigen BB, 10mM EDTA) with protease inhibitors, and chromatin was sheared using the Bioruptor (Diagenode). Lysate was diluted with 750μl dilution buffer (20mM Tris/HCl, 100mM NaCl, 0.5% TritonX-100, 2mM EDTA), 1% was taken as input DNA, and immunoprecipitation was carried out overnight with Dynabeads protein G bound to specific antibodies for PU.1 (Santa Cruz, sc- 352X), C/EBP (Santa Cruz, sc-150), H3K4me2 (Millipore, 07-030), H3K27ac (Active Motif, 39135), CJUN (Santa Cruz, sc-1694), P65 (Santa Cruz, sc-372X), USF2 (Santa Cruz, sc-862X) and RUNX1 (Santa Cruz, sc-365644). Beads were washed twice each with wash buffer I (20mM Tris/HCl, 150mM NaCl, 0.1% SDS, 1% Triton X-100, 2mM EDTA), wash buffer II (10mM Tris/HCl, 250mM LiCl, 1% IGEPAL CA-630, 0.7% Na-deoxycholate, 1mM EDTA), TE 0.2% Triton X-100 and TE 50mM NaCl and subsequently eluted with elution buffer (TE, 2% SDS). DNA was reverse- crosslinked and purified using ChIP DNA Clean & Concentrator (Zymo Research) according to the manufacturer's instructions. Sequencing libraries were prepared from eluted DNA by blunting, A-tailing, adaptor ligation as previously described (Heinz et al., 2010) using NextFlex barcodes (Bioo Scientific). Libraries were PCR-amplified for 12-15 cycles, size selected using PAGE/TBE gels for 200-400bp fragments by gel extraction and single-end sequenced on HiSeq 4000 or NextSeq 500.
attributes.tag: GEO Accession
attributes.value: GSM2974758