GSM8502702: CT3-EVTd3_GCM1_rep2; Homo sapiens; ChIP-Seq
Cell samples are fixed with 0.66% paraformaldehyde (FisherSci) diluted with PBS and incubated on a rotator for 10 minutes at room temperature. Samples are quenched by adding glycine to a final concentration of 0.125M with continued rotation for another 10 minutes at room temperature then wash with cold PBS 2x and replaced with PBS + 1mM EDTA + protease inhibitor (Roche)before collection with a cell scrapper. Cell pellets were centrifuged at 13200 rpm for 5 mins. Nuclear lysis extraction begins with 10 min incubation on a rotator in nuclear lysis buffer (50mM HEPES pH 7.8, 0.5% Triton X-100, 1mM EDTA, 0.5mM EGTA, 140 mM NaCl, 10% glycerol and 1% NP-40). Nuclear pellets are collected by centrifugation at 13200 rpm for 5 minutes at 4°C. Nuclei are resuspended in nuclear wash buffer (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, and 0.5mM EGTA) and then rotated 5 minutes at 4°C the repeated twice. Nuclear pellets are then resuspended in SDS lysis buffer (50mM Tris-HCl pH 8, 10mM EDTA, 1% SDS, and 1% Triton X-100). Nuclei samples are transferred to a 1ml tube (Bioruptor) and keep cold before sonication. Sonication was performed using the Diagenode Bioruptor sonicator (settings: 30sec on, 30 sec off, 30-35 cycles). Dilute the sonicate by 10-fold with Dilution buffer (25mM Tris-HCl pH 8, 150 mM NaCl, 3mM EDTA, and 1% Triton X-100). Pre-washed Protein G Sepharose beads (SIGMA, P3296-5ml) are prepared by washing them three times with dilution buffer. A pre-clearing step is performed by using 40μl of pre-washed beads combined sonicated sample. This rotated for 1hrs at 4°C then centrifuged at 1000xg for 1 min. Sticky-chromatin is remove by the beads and the supernatant is retained for the IP. 2ug of GCM1(HPA001343) antibody is added to each pre-cleared sample and then rotated overnight at 4°C. The next day, pre-washed Protein G Sepharose beads are added to the ChIP samples and rotated 2 hours at 4°C. The beads washed with 3 buffer: DB150 (25mM Tris-HCl pH 8.0, 150mM NaCl, 3mM EDTA, 1% Triton X-100, and 0.05% SDS), DB500 (25mM Tris-HCl pH 8.0, 500mM NaCl, 3mM EDTA, 1% Triton X-100, and 0.05% SDS), Buffer III (10mM Tris-HCl pH 8.0, 250mM LiCl, 1% Sodium Deoxycholate, 1% NP-40, and 1mM EDTA, and lastly TE buffer (10mM Tris-HCl pH 8.0, 1mM EDTA). DNA is eluted for 15 min on shaker rotating at 1000rpm with 200ul elution buffer (100 mM NaHCO3, and 1% SDS). 16ul of 5M NaCl is added to each sample and further incubated at 65C overnight for decrosslinking. Ethanol precipitation is performed to collect ChIP material, and further purification was performed with Geneaid Gel/PCR cleanup protocol. Purified DNA fragments passed to sequencing library preparation. Up to 5ng of isolated genomic DNA were re-sonicated to generate fragment ~100bp. Libraries were prepared using and following the NEBNext Ultra II DNA Library Prep with Sample Purification Beads protocol.