GSM5114303: GATA3_HCC1954_1; Homo sapiens; ChIP-Seq
Cells were cross-linked with 1% formaldehyde for 8 minutes and quenched by 125mM glycine for 5 minutes at room temperature with gentle shaking. Cells were quickly rinsed in cold PBS twice then scraped in 5mL cold PBS on ice and collected in a 15mL conical tube. Cells were centrifuged at 4°C at 1250xg for 3 minutes. Cell pellets were rinsed with 5mL cold PBS, centrifuged at 4°C at 1250xg for 3 minutes and snap-frozen in liquid nitrogen. Cell pellets were thawed on ice and resuspended in 1mL lysis buffer 1 (50mM HEPES-KOH pH 7.5 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100) supplemented with protease inhibitors and incubated at 4°C on a rocker for 10 minutes. Lysates were centrifuged at 1000 rpm at 4°C for 5 minutes. Pellets were resuspended with 1mL lysis buffer 2 (10mM Tris pH 8.0 1mM EDTA 0.5mM EGTA 200mM NaCl) supplemented with protease inhibitors and incubated at 4°C on a rocker for 10 minutes. Lysates were centrifuged at 1000 rpm at 4°C for 5 minutes. Pellets were resuspended in 1mL shearing buffer (0.1% SDS, 1mM EDTA, 10mM Tris HCl pH 8.0) supplemented with protease inhibitors, then centrifuged at 1000 rpm at 4°C for 5 minutes. Pellets were resuspended in 500µL shearing buffer, transferred in a 1mL covaris milliTUBE and sonicated with a Covaris E220 sonicator for 8 minutes with 5% duty, 140 peak incident power and 200 cycles per burst. The sonicated lysates were centrifuged at 16000xg for 15 min at 4°C to pellet cellular debris. Sonicated chromatin in the supernatant was transferred to a new 1.5 ml LoBind Eppendorf tube. Immunoprecipitation and elution were performed using the ChIP- IT High Sensitivity kit (Active Motif #53040) according to the manufacturer's instructions. ChIP-seq libraries (GenomEast platform) were prepared from 3 to 10 ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v2 (C05010014, Diagenode s.a., Seraing, Belgium), according to manufacturer's instructions. In the first step, the DNA was repaired and yielded molecules with blunt ends. In the next step, stem-loop adaptors with blocked 5 prime ends were ligated to the 5 prime end of the genomic DNA, leaving a nick at the 3 prime end. The adaptors cannot ligate to each other and do not have single-strand tails, avoiding non-specific background. In the final step, the 3 prime ends of the genomic DNA were extended to complete library synthesis and Illumina compatible indexes were added through a PCR amplification (7 + (0-2) cycles). Amplified libraries were purified and size-selected using Agencourt AMPure XP beads (Beckman Coulter) to remove unincorporated primers and other reagents.