GSM6194034: prEpiSCs_H3K4me3; Homo sapiens; ChIP-Seq
5×10^6 prEpiSCs were fixed in fresh 1% formalin/prEpiSC medium (v/v) for 10 min followed by quenching with glycine (125 mM)  for 5 minutes at room temperature. Fixed cells were washed twice in ice-cold PBS, re-suspended in Lysis buffer 1 (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5%NP-40, 0.25% Triton X-100 and protease inhibitors) and incubated on ice for 10 minutes. Cells were then pelleted by centrifugation and resuspended in Lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and protease inhibitors) with incubation on ice for 10 minutes. Cells were then pelleted and resuspended in 0.25ml of freshly prepared Lysis buffer 3 (10 mM Tris-HCl, pH 8.0,100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5%N-lauroylsarcosine and protease inhibitors) and sonicated to about 100 - 500 bp fragments using a Bioruptor (Diagenode). Anti-H3K4me3 antibody was  incubated with preblocked protein A magnetic beads (Invitrogen) at 4°C with gentle agitation on a rotator for 4 hours. Antibody-bead complexes were added into the sonicated chromatin solution and rotated for overnight at 4°C. Chromatin-bead immunoprecipitates were collected by the magnetic device and washed sequentially with high-salt wash buffer (20 mMTris·HCl, pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, and 1% Triton X-100), then with LiCl wash buffer (10 mM Tris·HCl, pH 8.0, 250 mM LiCl, 1 mM EDTA, and 1% Nonidet P-40), and then with Tris-EDTA (TE)-NaCl buffer (1 mM EDTA, 10 mM Tris·HCl, pH 8.0, and 50 mM NaCl). Immunoprecipitated DNA was eluted by incubating the beads with 100 µl elution buffer (50 mM Tris·HCl, pH 8.0, 10 mM EDTA, and 1% SDS), shaking at a setting of 1300 rpm for one hour at room temperature. For reverse crosslinking, sample was treated with proteinase K and incubated at 65°C for 4 hours. The DNA was purified using phenol-chloroform extraction, and suspended in 40 µl TE buffer. ChIP-seq library was constructed with the Accel-NGS 2S Plus DNA Library kit (Swift Biosciences, catalog. no. 21096) according to the manufacturer's instructions. The libraries were sequenced on an Illumina Hiseq platform and 100 bp single-end reads were generated.