ChIP-seq for FOXA1, ER and CTCF in breast cancer cell lines
ChIP-seq for FOXA1, ER and CTCF in breast cancer cell lines
RPMI 1640 medium por DMEM supplemented with 10% inactivated FBS, l-glutamine and PEST at 37C with 5% CO2. Antibodies were from Santa Cruz Biotechnology (sc-543), abcam (ab5089 and ab23738) and Millipore (07-729) and ChIP was performed using 10e7-10e8 cells per reaction. Briefly, crosslinking of protein and DNA was done in room temperature for 10 minutes with serum free media containing 0,37 % formaldehyde. Cells were scraped and washed in PBS before treatment of cell lysis buffer with protease inhibitors on ice. Pelleted nuclei were resuspeded in RIPA buffer and a BioRuptor (Diagenode) was used to sonicate DNA. ChIP was performed with 10 micrograms of antibody. ChIP DNA was gel fractionated and amplified to create two size libraries corresponding to insert sizes of 200-300 bp. For DNase I hypersensitivity was performed as previously described (Eeckhoute et al., 2006). Digested DNA was run on a gel and the digested material (between 200bp and 500bp in size). For all ChIPs washing was done six times with RIPA buffer and once with TE-buffer and DNA-protein complexes were eluted from beads twice with 200 microlitres of 0.1 M NaHCO3 and 1 % SDS and treated with RNAseA at 65C for 6 hours and Proteinase K at 45C over night. Chromatin was amplified with Solexa protocol chromatin amplification.
Experimental Factor: CELL_LINE
TAM_R
Experimental Factor: IMMUNOPRECIPITATE
ER