The human neuroblastoma cell line SK-N-SH (ATCC: HTB-11) was grown at 37°C in a 5% CO2 environment in DMEM (Invitrogen Corp.) supplemented with 1% Glutamax (Invitrogen), 10% foetal bovine serum (Wisent), 100 IU/ml penicillin and 100 μg/ml streptomycin (Invitrogen).
Extracted molecule
genomic DNA
Extraction protocol
Cells were treated with 1% formaldehyde for 8 min to crosslink proteins to DNA. Glycine (0.125 M) was added to quench the reaction. Cells were collected using a cell scraper, washed twice in cold PBS1X, washed for 10 min in solution I (10 mM HEPES, pH 7.5, 10 mM EDTA, 0.5 mM EGTA, 0.75% Triton X-100) and 10 min in solution II (10 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA). Cells were resuspended in lysis buffer (25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) and sonicated for 6 times 10 s (30% output) to shear chromatin to an average size of 0.5 kb using a Fisher Sonic Dismembrator sonicator.
The human neuroblastoma cell line SK-N-SH (ATCC: HTB-11) was grown at 37°C in a 5% CO2 environment in DMEM (Invitrogen Corp.) supplemented with 1% Glutamax (Invitrogen), 10% foetal bovine serum (Wisent), 100 IU/ml penicillin and 100 μg/ml streptomycin (Invitrogen).
Extracted molecule
genomic DNA
Extraction protocol
Cells were treated with 1% formaldehyde for 8 min to crosslink proteins to DNA. Glycine (0.125 M) was added to quench the reaction. Cells were collected using a cell scraper, washed twice in cold PBS1X, washed for 10 min in solution I (10 mM HEPES, pH 7.5, 10 mM EDTA, 0.5 mM EGTA, 0.75% Triton X-100) and 10 min in solution II (10 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA). Cells were resuspended in lysis buffer (25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) and sonicated for 6 times 10 s (30% output) to shear chromatin to an average size of 0.5 kb using a Fisher Sonic Dismembrator sonicator.
Raw data were first background subtracted and normalized using median normalization. Regions significantly enriched for PARP3 relative to IgG were identified as described (Lee et al. 2006). Briefly, the genome was scanned three probes at a time. When three probes were in a window of 1000 bp or less, they were further refered to as triplets. Triplets were considered significant when the PARP3 signal in 2 out of 3 probes had a p-value lower than 0.05 or when the center probe signal had a p-value lower than 0.01 and the first and last probes had a p-value lower than 0.1. The false discovery rate is estimated at 0.005%. All the data were process within R using the limma package.
