title: GSM8901898: H3K27ac ChIP-seq in iGMPs Replicate 1; Mus musculus; ChIP-Seq
library_construction_protocol: H3K27ac ChIP-seq was performed as previously described (Franklin et al. 2022). For each ChIP, 10 million iGMPs were fixed in culture media with 4% formaldehyde for 15 minutes at room temperature. After fixing, cells were washed with DPBS and the cell pellet was flash frozen in liquid nitrogen and stored at -80 °C. Nuclei were isolated by lysing cells with cell lysis buffer (20mM Tris-HCl pH 8.0, 85mM KCl, 0.5% NP40) for 10 minutes at 4 °C and spinning at 12,000 RPM for 4 min at 4 °C. Nuclei were then lysed for 10 min at 4 °C in nuclear lysis buffer (10mM Tris-HCl pH7.5, 1% NP40, 0.5% Na Deoxycholate, 0.1%SDS). Lysed nuclei were sonicated using a Branson 250 probe sonifier at 40% amplitude for 5 minutes in an ice bath to garner 50% of fragments below 1000 bp, and less than 10% under 200 bp. Chromatin was stored at -80 °C while confirming input fragment distribution by gel electrophoresis or TapeStation. Sheared chromatin was supplemented with 167 mM NaCl and 1.2mM EDTA for immunoprecipitation. Protein A or G beads (for rabbit or mouse antibodies, respectively; 10002D/10004D, ThermoFisher) pre-conjugated with 2 µg of antibody were added to the sheared chromatin for 2 hours at 4 °C for each target. The following antibodies were used: H3K27ac (ab4729, Abcam). Immunoprecipitations were washed sequentially two times each with low salt buffer (0.1% DOC, 0.1% SDS, 1% Triton X-100, 140 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl pH 8.0), high salt buffer (0.1% DOC, 0.1%SDS, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl pH 8.0), LiCl buffer (0.25 M LiCl, 1% NP40, 1% Na Deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0), and TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) and then eluted in elution buffer (10 mM Tris-HCl pH 8.0, 5 mM EDTA, 300 mM NaCl, 0.1% SDS). Immunoprecipitations were reverse crosslinked overnight at 65 °C after adding 7.25x reverse crosslinking buffer (250 mM Tris-HCl pH 6.5, 1.25 M NaCl, 62.5 mM EDTA, 5 mg/ml Proteinase K [EO0491, ThermoFisher], 62.5 ug/ml RNase A [EN0531, ThermoFisher]). Input samples were reverse crosslinked by diluting sheared chromatin 1:5 with water and adding 7.25x reverse crosslinking buffer overnight at 65 °C. Reverse-crosslinked DNA was isolated with 1.8x AMPure XP (A63880, Beckman Coulter) beads and used for library preparation. NEB Ultra II DNA Library Prep and Index Kits (E7645, E6440) were used according to the manufacturer's protocol with modifications. Briefly, up to 10 ng DNA was used for end preparation and adapter ligation without size selection. NEB indexes were added with 2-step PCR using an optimized number of cycles (10-15) and reduced extension times (15 s). Fragment distribution of prepared libraries were confirmed by Bioanalyzer to contain over 90% fragments in the 200-600 bp range and then submitted to Illumina NovaSeq 6000 paired-end sequencing at the University of California, Irvine Genomics Research and Technology Hub.
identifiers.namespace: GEO
identifiers.id: GSM8901898_r1