GSM3462430: Input_B; Homo sapiens; ChIP-Seq
Cells were washed with 10 ml of intact RPMI1640 media and fixed with 10 ml of fresh 1% paraformaldehyde in RPMI1640 media for 15 min at room temperature with gentle shaking. The fixation was quenched by adding 600 ul of 2.5 M glycine and incubating at room temperature for 5 min. The fixed cells were scraped and then resuspended after adding 1 ml of lysis H buffer (150 mM NaCl, 50 mM HEPES (7.5), 5 mM EDTA, 0.5% (vol/vol) IGEPAL CA-630, 1.0% (vol/vol) Triton-X100). The cells were collected with centrifugation in a 1.5 ml tube/flask at 2 krpm for 5 min at 4°C and washed twice with 500 ul of the cell wash buffer (1/10 diluted Lysis H buffer by HBSS). Resulting cell pellet was stored at -80°C until following analysis (one sample results in two flasks and then two tubes of cell pellets). One tube of cell pellet was washed twice with 1 ml of the lysis H buffer with protease inhibitors and collected with centrifuge. The cell pellet was resuspended with 1.5 ml of 1% SDS in the lysis H buffer. An aliquot (500 ul) of the cell suspension was transferred to a 1.5 ml tube to perform sonication by the BRANSON digital Sonifier (power 40%; pulse ON, 0.7 sec.; interval 1.3 sec.; total 30 min). The resulting lysates were cleared by twice of centrifuges and pooled. For input DNA control, 50 ul of the cleared lysate was saved. All of the left lysate was diluted with 13.5 ml of lysis H buffer with inhibitors, and this was incubated with 90 ul of washed suspension of the Streptavidin sepharose resin at 4°C for one overnight with gentle shacking. The resin was washed with once with 1 ml of the wash buffer I (20 mM Tris-HCl (8.0), 150 mM NaCl, 2mM EDTA, 1% TritonX-100, 0.1% SDS), II (20 mM Tris-HCl (8.0), 500 mM NaCl, 2mM EDTA, 1% TritonX-100, 0.1% SDS), III (20 mM Tris-HCl (8.0), 1mM EDTA, 0.5% Na deoxycholate, 1% IGEPAL), and twice with 1 ml of 100 mM Tris-HCl (pH 8.0), 10 mM EDTA, and then eluted with 90 ul of TESK buffer (25 mM Tris-HCl (pH8.0), 1 mM EDTA, 1% SDS, 20 ug/ml Protease K) by incubation at 55°C for 30 min. After recovering the first elution fraction, the remaining DNAs were further eluted with additional 90 ul of TESK buffer with incubation at 65°C for one overnight. The first elution fraction was also incubated at 65°C for one overnight. Pooled elution solutions (total about 180 ul) was treated with 3.6 ul of 10 mg/ml RNaseA at 37°C for 1 hour and then 9 ul of 10 mg/ml Protease K with presence of 1 mM CaCl2 at 55°C for 1 hour. The resulting solution was subjected to DNA purification by the MinElute PCR kit (Qiagen) with twice of 10 ul elution (total 20 ul). Libraries were prepared according to Illumina's instructions (Prep kit IP-202-1012)