GSM8384298: ES cells_IgG_wt_rep1; Mus musculus; OTHER
Pellets were resuspended in BioMag Plus Concanavalin A beads (Bangs Laboratories), equilibrated in Binding Buffer with 20 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2 and1 mM MnCl2. Cell pellets were resuspended in Wash Buffer, mixed with a slurry of equilibrated Concavalin A coated magnetic beads, and rotated for 10 mins at room temperature. For each sample, 10 µl bead slurry was used and were placed on a magnetic separator to discard the supernatant. Beads were again resuspended in Wash Buffer containing 2 mM EDTA, 0.1% bovine serum albumin, 0.05% Digitonin, and 1:50 dilution of primary antibody against H3K27Ac (Abcam, ab4729). This was incubated on a nutating platform for 2 hours at room temperature. After incubation, beads were washed twice in Digitonin Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche Complete Protease Inhibitor no EDTA, 0.05% Digitonin and 0.1% bovine serum albumin), then incubated with lab prepared pA-MNase (600 µg/ml, 1:200) in Digitonin Buffer for 1 hour at 4 °C. After incubation, beads were washed twice, resuspended in 150 µl of Digitonin Buffer, and equilibrated to 0°C before adding 2mM CaCl2 and incubated at 0°C for 1 hour. After incubation, 150 µl of 2X Stop Buffer containing 200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/ml RNase A and 40 µg/ml glycogen was added. Beads were incubated for 30 mins at 37°C and then spun at 16,000 g for 5 mins at 4°C. Supernatant was transferred, mixed with 3 µl 10% SDS and 1.8U Proteinase K (NEB #P8107S) and incubated overnight at 55°C, shaking at 900 rpm. After incubation, 300 µl of 25:24:1 Phenol/Chloroform/ Isoamyl Alcohol was added, solutions were vortexed, and transferred to Maxtrack phase-lock tubes (Qiagen #129046). Tubes were centrifuged at 16,000 g for 3 mins at room temperature. 300 µl of Chloroform was added, solutions were mixed by inversion, and centrifuged at 16,000 g for 3 mins at room temperature. Aqueous layers were transferred to new tubes and DNA was isolated by ethanol precipitation and resuspended in 10 mM Tris-HCl pH 8.0 (Thermo Fisher #15568025). Cut&Run libraries were prepared following the SMARTer ThruPlex TAKARA Library Prep kit with small modifications. For each sample, 10 µl of double stranded DNA, 2 µl of Template Preparation D Buffer and 1 µl of Template Preparation D Enzyme were combined, and End Repair and A-tailing was performed in a thermocycler with a heated lid at 22°C, for 25 mins and 55°C for 20 mins. 1 µl each of Library Synthesis D Buffer and Library Synthesis D Enzyme were subsequently added, and library synthesis was performed at 22 °C for 40 mins. Immediately after, 25 µl of Library Amplification D Buffer, 1 µl of Library Amplification D Enzyme, 4 µl of nuclease-free water and 5 µl of unique Illumina-compatible indexed primer were added to each sample. Library amplification was performed using the following cycling conditions: For denaturation - 72°C for 3 mins, 85°C for 2 mins and 98°C for 2 mins, addition of unique indexes - 4 cycles of 98°C for 20 secs, 67°C for 20 secs and 72°C for 10 secs, library amplification - 14 cycles of 98°C for 20 secs and 72°C for 10 secs. Post-PCR clean-up was performed on amplified libraries with SPRI beads using 0.6X left/1x right double size selection, washed twice gently in 80% ethanol and eluted in 10–12 µl 10 mM Tris pH 8.0. Paired end 50