title: GSM2454344: mESC_Inp_parental_rep1_UNT; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP sequencing for H3K4me3 in mouse ES cells was performed by calibrated native ChIP sequencing. Calibrated ChIP-seq was carried out in biological triplicate for each cell line and condition. This was achieved by adding 2.5x10^6 Drosophila S2 (SG4) cells to 10x10^6 untreated or tamoxifen treated Cfp1 fl/fl cells. Nuclei were released by resuspending the mixed cell mixture in ice cold lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40). Nuclei were then washed, and resuspended in 1 ml (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.25 M sucrose, 3 mM CaCl2, 1x protease inhibitors, and incubated with 100U of MNase (Fermentas) at 37°C for 5 min followed by the addition of EDTA to halt the digestion. The supernatant was collected following centrifugation at 1500 g for 5 min at 4°C. The remaining pellet was incubated with 300 ul of nucleosome release buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2 mM EDTA, 1x PIC) at 4°C for 1 h, then passed through a 27G needle using a 1 ml syringe, and spun at 1500 g for 5 min at 4°C. The two supernatants were combined. For each immunoprecipitation 20 µl of IPA300 agarose beads (RepliGen) were blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA in native ChIP incubation buffer (70 mM NaCl, 10 mM Tris-HCl pH 7.5, 2 mM MgCl2, 2 mM EDTA, 0.1% Triton X-100). Native chromatin was then diluted 10-fold in (70 mM NaCl, 10 mM Tris-HCl pH 7.5, 2 mM MgCl2, 2 mM EDTA, 0.1% TritonX-100, 1x PIC) and incubated with 20 µl of blocked beads per 1 ml of diluted chromatin at 4°C for 1 h. Beads were washed four times with native ChIP wash buffer (20 mM Tris-HCl pH 7.5, 2 mM EDTA, 125 mM NaCl, 0.1% Triton-X100), and once with 1 ml ice cold TE buffer. DNA was purified with the ChIP DNA Clean & Concentrator kit (Zymo Research). For CFP1 and T7-SET1A ChIP-seq in ES cells, an equal number of untreated and treated cells were resuspended in 10 mL of PBS and subjected to crosslink with 1% methanol-free formaldehyde (ThermoFisher) for 10 min at 25°C and quenched with 150 mM glycine for 10 min at RT. For T7-SET1A ChIP, cells were first crosslinked in 2 uM disuccinimidyl glutarate (DSG, ThermoFisher) at 25° C for 50 mins and then 1% formaldehyde was added for 10 min at 25°C. Cells were lysed on ice for 10 min in 1 mL of lysis buffer (50 mM HEPES pH7.9, 300 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.5% NP-40, 0.1% Na-deoxycholate, 0.1% SDS, 1x Complete EDTA-free inhibitor cocktail, 1 mM AEBSF) and then sonicated with Bioruptor Pico (Diagenode) for 20 min with 30s ON/OFF cycles (for double crosslinked chromatin, cells were sonicated for 23 min). Lysates were centrifuged for 10 min at 16000 g and the cleared chromatin was recovered and DNA quantified. For each IP, 300 ug of chromatin was diluted to 1 mL of lysis buffer and pre-cleared for 1 h at 4°C with IPA 300 resin beads (Repligen) blocked with yeast tRNA and BSA. Antibodies were then added to each IP and incubated overnight, followed by isolation of antibody-chromatin complexes with 40 uL of 50% blocked slurry beads per IP. Immunoprecipitates were washed 1x with lysis buffer, 1x with lysis buffer at 500 mM NaCl, 1x DOC buffer (10 mM Tris-HCl pH 8, 250 mM LiCl, 1 mM EDTA, 0.5% NP40, 0.5% Na-deoxycholate), 2x with TE pH 8. Chromatin was then eluted in 200 uL of elution buffer (1% SDS, 0.1 M NaHCO3) for 30 min at 30°C with vigorous shaking. Eluates and inputs were treated with DNase-free RNase (ThermoFisher) and crosslinks were reversed at 65 ˚C overnight with 200 mM NaCl and ProteinaseK solution (Sigma). DNA was purified with the ChIP DNA Clean & Concentrator kit (Zymo Research). For massively parallel sequencing, DNAs were post-sonicated with Bioruptor Pico (Diagenode) to a DNA fragment size of 200-300 bp as determined by Bioanalyser analysis. Libraries were prepared with NEBNext Ultra DNA Library Prep Kit or with NEBNext EndPrep Dual Index for H3K4me3 ChIP-seq and quantified by qPCR using KAPA Illumina DNA standards as reference. Libraries were sequenced on an Illumina NextSeq500 platform.