title: GSM5213103: BMDM_p65_2OHE2_LPS_ChIP; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were washed 2x with PBS and fixed with 0.67mg/mL DSG (Thermo Fisher) in PBS for 30 minutes at room temperature with shaking, followed by addition of paraformaldehyde (Electron Microscopy Sciences) to 1% and an additional 15 minutes of room temperature fixation. Fixation was quenched by adding glycine to 125mM and shaking 10 minutes, after which fixed cells were scraped, pooling three independent 15cm plates per ChIP condition (approximately 3.0x10^7 cells per ChIP). Cell pellets were washed 2x with PBS, resuspended in 1.5mL ice cold ChIP RIPA buffer (20mM Tris HCl pH 8.0, 150mM NaCl, 2mM EDTA, 0.1% SDS, 1% Triton X-100) with protease inhibitors (Roche), and sonicated with metal probe adaptor using Bioruptor (Diagenode) for 60 minutes (continuous cycles of 20 seconds ON/40 seconds OFF, “medium” setting). Samples were spun 20 minutes at maximum speed at 4ºC in a benchtop centrifuge to remove insoluble material, and supernatant containing soluble sheared chromatin transferred to new tube, saving 1% volume for input library preparation. Immunoprecipitation was performed overnight at 4ºC with 2mg of primary antibody (p65: Santa Cruz sc-372, H3K27ac: Abcam ab4729), or corresponding species-appropriate IgG control antibody (GenScript), conjugated to Protein A Dynabeads (Invitrogen). The next day, beads were captured with magnet on ice and washed with ice cold wash buffer II (20mM Tris HCl pH 8.0, 150mM NaCl, 2mM EDTA, 1% Triton X-100, 0.5% NaDOC) 3 times, wash buffer III (10mM Tris HCl pH 8.0, 250mM LiCl, 2mM EDTA, 1% NP-40, 0.5% NaDOC) 3 times, and TE with 50mM NaCl two times, all supplemented with protease inhibitors. Beads with antibody-target-DNA complexes were then resuspended in 200mL elution buffer (50mM Tris HCl pH 8.0, 10mM EDTA, 1% SDS) for 30 minutes at 37ºC to elute immunoprecipitated complexes. 200mL eluent was collected, 10mL of 5M NaCl added, and DNA-protein crosslinks reversed overnight at 65ºC. Next day, samples were treated with RNAse A (Thermo Fisher) for 1 hour at 37ºC, Proteinase K (NEB) for 1 hour at 50ºC, and DNA fragments recovered with Zymo ChIP DNA Clean & Concentrator kit. Sequencing libraries were prepared from both input chromatin, and chromatin recovered from immunoprecipitations, using in-house protocol. Briefly, DNA was blunted, A-tailed, and ligated to Illumina-compatible NEXTFlex sequencing adaptors (Bioo). Libraries were PCR amplified, gel size selected, quantified, pooled, and sequenced on an Illumina HiSeq2500.
attributes.tag: GEO Accession
attributes.value: GSM5213103