GSM2545828: TCF4_ChIP_rep1; Homo sapiens; ChIP-Seq
Confluent cells were fixed with 1% (v/v) formaldehyde for 5min at room temperature and then quenched with 125mM glycine. Cells were then washed twice with cold phosphate buffered saline (PBS) and collected by scraping into cold PBS. Cells were pelleted by centrifugation (2000g, 5min, 4°C) and then washed in PBS and collected by centrifugation as before. 1 x 107 pelleted cells were lysed in 1ml ChIP buffer (150mM NaCl, 50mM Tris-HCl (pH 7.5), 5mM EDTA, NP-40 (0.5% v/v), Triton X-100 (1.0% v/v) for 30 min on ice. Nuclei were released by passing the lysate through a 23-gauge needle. Nuclei were collected by centrifugation (2000g, 5 min, 4°C), the supernatant was discarded and remaining pellet resuspended in 1ml of shearing buffer (50mM Tris pH 8.0, 5mM EDTA, 150mM NaCl, 0.1% (v/v) SDS). The lysate was then clarified by centrifugation (13,000rpm for 10min at 4°C) and the supernatant transferred to a new tube. Chromatin was fragmented by sonication using a Covaris S2 at recommended settings. Following sonication, SDS was sequestered by addition of Triton-X-100 to a final concentration of 1% (v/v). Sonicated chromatin was then pre-cleared for 1h at 4°C using bovine serum albumin (BSA) blocked beads.     For each ChIP reaction, chromatin prepared from approximately 1 x 107 cells was incubated with 5µg anti-TCF4 or anti-IgG antibody, overnight at 4°C with rotation. The chromatin was then centrifuged (13,000rpm for 10min at 4°C) and the supernatant transferred to a tube containing BSA-blocked beads. Protein-antibody complexes were then captured for 30 min at 4°C with rotation. Beads were then washed twice with ChIP buffer containing 0.5M NaCl, followed by five washed with LiCl wash buffer (50mM Tris pH 8.0, 5mM EDTA, 0.5% (v/v) NP-40, 1.0% (v/v) Triton X-100, 500mM LiCl and then twice with rinse buffer (50mM Tris pH 8.0, 5mM EDTA). Beads were then resuspended in 100µl elution buffer (10mM Tris pH 8.0, 1mM EDTA, 0.1% (w/v) SDS) and incubated with RNase for 30 min at 37°C). Proteinase K was added and incubated over night at 60°C. The eluate was then cleaned up using the QIAgen PCR clean up Kit (Qiagen). Enriched chromatin was processed and sequenced by Source BioScience (Nottingham, UK). using the Illumina TruSeq ChIP Sample Preparation Kit (San Diego CA, USA). Barcode info: TCF4_rep1 = GTGAAA, TCF4_rep2 = CAGATC, IgG_rep1 = ACAGTG, IgG_rep2 = GCCAAT