GSM5065883: SGCAS Input rep1 [ChIP-seq]; Mus musculus; ChIP-Seq
For each ChIP, 5×10^6 cells were used. Cells were fixed in 1% formalin/mESC medium (v/v) for 10 min with gentle agitation on a rotator at room temperature. Fixation was stopped by the addition of glycine (125 mM) and agitation for 5 minutes at room temperature. Fixed cells were washed twice in ice-cold PBS, re-suspended in LB1 (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). After 10 min of incubation, cells were pelleted by centrifugation and resuspended in LB2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA and protease inhibitors). After 10 min of incubation, cells were pelleted and resuspended in 0.25ml of freshly prepared LB3 (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-H3K79me2 and anti-H3K79me3 antibodies were diluted into 5 mg/ml BSA (PBS/BSA) and incubated with preblocked protein G magnetic beads (Invitrogen) at 4°C with gentle agitation on a rotator for 4 hours. Antibody-bead complexes were rotated overnight with sonicated chromatin solution at 4°C. Immune chromatin-bead precipitates were collected by the magnetic device (Invitrogen). Precipitates were 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 room temperature at a setting of 1300 rpm for one hour. To reverse crosslinking, sample was treated with proteinase K and incubated at 65°C for 4 hours. Add 100 µl TE buffer to each sample. 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 150 bp paired-end reads were generated.