title: Carroll_ChIP_exo
design: Carroll_ChIP_exo
library_construction_protocol: The MCF-7, MDA-MB-231, MDA-MB-453, LNCaP and ZR-75-1 cell lines were obtained from American Type Culture Collection (ATCC). Cells were grown in DMEM (MCF-7, MDA-MB-231 and MDA-MB-453) or RPMI (LNCaP and ZR-75-1), both supplemented with 10% Fetal Bovine Serum, 2 mM L-glutamine, 50U/ml penicillin and 50ug/ml streptomycin in 37_C incubator with 5% CO2. For all experiments, 1x10^8 cells were cross-linked with 1% formaldehyde as previously described (Schmidt et al., Methods 48(3) July 2009 doi:10.1016/j.ymeth.2009.03.001). The ChIP assays were performed as previously described (Schmidt et al., 2009). Protein-bound DNA was immunoprecipitated with antibodies against COT2/COUP-TFII (R&D Systems, PP-H7147-00), FoxA1 (Abcam, ab5089), ER (Santa-Cruz, sc-543), FoxA2 (Santa-Cruz, sc-6554) and P300 (Santa-Cruz, sc-585).  For the ChIP-exo library preparation, the immunoprecipitated chromatin was end-repaired, ligated to the TruSeq P7 exo-adapter, nick repaired, digested with the lambda and the RecJf exonucleases. The digested DNA was extracted, second-strand synthesised with the P7 primer, ligated to the TruSeq P5 exo-adapter and finally amplified with 18 PCR cycles. A 2% agarose gel was used to select 200-300 bp DNA fragments.
attributes.tag: Experimental Factor: cell line
attributes.value: MCF-7
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: COT2
attributes.tag: Experimental Factor: organism
attributes.value: Homo sapiens
attributes.tag: Experimental Factor: protocol
attributes.value: ChIP-exo