GSM8724811: HEK293_pThr4_Mut_rep2; Homo sapiens; ChIP-Seq
Crosslinking was performed using 1% formaldehyde for 10 min. Crosslinking was quenched with 0.125 M glycine for 5 min. Cells were successively lysed in lysis buffer 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, 1× PI), LB2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1× PI) and 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, 1× PI). Chromatin was sonicated to an average size of ~200–500 bp using UCD-200 Biorupter (30s on and 30 s off for 30 min). A total of 5 μg of pThr4 antibody (Active Motif, cat: 61361) was pre-mixed in a 50μl volume of Dynabeads protein A or protein G (Invitrogen) and was added to each sonicated chromatin sample and incubated overnight at 4°C. The chromatin-bound beads were washed two times with low salt buffer (0.1% Na Deoxycholate, 1% Triton X-100, 1mM EDTA, 50mM HEPES pH 7.5, 150mM NaCl), once with high salt wash buffer (0.1% Na Deoxycholate, 1% Triton X-100, 1mM EDTA, 50mM HEPES pH 7.5, 500mM NaCl), once with LiCl wash buffer (250mM LiCl, 0.5% NP-40, 0.5% Na-Deoxycholate, 1mM EDTA, 10mM Tris-Cl pH 8.0) and twice in TE buffer. The chromatin was reverse crosslinked overnight at 65°C with shaking at 750rpm. After DNA extraction using phenol-chloroform, the DNA was resuspended in 10mM Tris-HCl pH 8.0. The extracted DNA was used to construct the ChIP-seq library using the NEBNext Ultra II DNA Library Prep Kit followed by sequencing with an Illumina HiSeq 3000.