GSM8579995: H3K27ac_mg_GrnKO_rep1_input; Mus musculus; ChIP-Seq
Brain nuclei were isolated as previously described (Nott et al., 2019,2021), with initial homogenization and crosslinking performed with 1% formaldehyde in Dulbecco's PBS for 10 minutes at room temperature. Nuclei were stained overnight with PU.1-PE (Cell Signaling 81886S), OLIG2-AF488 (Abcam 225099), and NEUN-AF488 (Millipore MAB 377X). Nuclei were stained with DAPI (BioLegend 422801) the next day then sorted with a BD Influx Cell Sorter. Nuclei were sorted as DAPI positive, singlets, PU.1+, OLIG2-, and NEUN-. Chromatin immunoprecipitation sequencing was performed as previously described (Heinz et al, 2010, Texari et al, 2021). For H3K27ac ChIP-seq, ~500,000 fixed nuclei were quickly thawed on ice and resuspended in ice-cold LB3 buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine) and 1x protease inhibitor cocktail. Nuclei / chromatin was sheared by sonication using the PIXUL Multi-Sample Sonicator (Active Motif) in low bind 96 well plates following the manufacturer's instructions. Samples were sonicated with the following settings: Pulse (N): 50, PRF [kHz]: 1.00, Process time: 60 min, Burst Rate [Hz]: 20.00. Samples were pulse spun to 600xg at 4°C in the 96-well plate, transferred to low-bind tubes, and chromatin recovered by retaining the supernatant following a spin at 21,000xg at 4°C for 10 minutes. The supernatant was then diluted 1.1-fold with ice-cold 10% Triton X-100. Two percent of the lysate was kept as ChIP input control. 10 μL of Dynabead Protein A and 10 μL of Protein G were added per sample, in addition to 2 μg of a specific antibody for H3K27ac (Active Motif 39685). The samples were rotated overnight at 4°C and were washed as follows the next day: 3x with Wash Buffer I (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100) + 1x protease inhibitor cocktail, 3x with Wash Buffer III (10 mM Tris-HCl pH 7.5, 250 mM LiCl, 1% Triton X-100, 1 mM EDTA, 0.7% Sodium Deoxycholate) + 1x protease inhibitor cocktail, 2x with TET (0.2% Tween-20/TE) + 1/3x protease inhibitor cocktail, and 1x with IDTET (0.2% Tween-20, 10 mM Tris pH 8, 0.1 mM EDTA). Samples were finally resuspended in 25 μL TT buffer (10 mM Tris pH 8 + 0.05% Tween 20) prior to on-bead library preparation. Sequencing libraries for H3K27ac ChIP and input samples were prepared with NEBNext Ultra II DNA library prep kit (NEB, E7645) reagents according to the manufacturer's protocol on beads suspended in 25 μL TT (10 mM Tris/HCl pH 7.5, 0.05% Tween-20), with reagent volumes reduced by half. DNA was eluted and crosslinks reversed by adding 4 μL 10% SDS, 4.5 μL 5 M NaCl, 3 μL EDTA, 4 μL EGTA, 1 μL proteinase K (20 mg/ml), 16 μL water, incubating for 1 h at 55°C, then 30 minutes to overnight at 65°C. DNA was purified using 2 μL of SpeedBeads (Cytiva GE45152105050250) in a 20% PEG8000 / 1.5M NaCl solution to final concentration of 12% PEG, then eluted with 25 μL TT. DNA contained in the eluate was then amplified for 12-14 cycles in 25 μL PCR reactions using NEBNext High-Fidelity Q5 2X PCR Master Mix (NEB) and 0.5 mM each of primers Solexa 1GA and Solexa 1GB. Resulting libraries were size selected by gel excision to 200-500 bp, purified, and paired-end sequenced using an Illumina NovaSeq instrument.