293T cells were reverse transfected with GFP or BRCA1 targeted siRNAs using Lipofectamine RNAiMax as per the manufacturers protocol. Following transfection, cells were grown for 72 hours in DMEM before fixing for ChIP.
Extracted molecule
genomic DNA
Extraction protocol
5x10E6 cells were fixed with 1.5% formaldehyde Chromatin was cross- linked using 1.5% formaldehyde for 15 minutes at room temperature. Cells were then washed twice with PBS and collected in 1 mL collection buffer [100mM Tris-HCL (pH 9.4) and 100mM DTT]. The cell suspension was then incubated on ice for 15 minutes. Cells were then lysed sequentially by resuspension and 5-minute centrifugation at 3,000g at 4oC with 1 mL buffer A (10mM EDTA, 0.5mM EGTA, 10mM HEPES, and 0.25% Triton X-100) and 1 mL buffer B (1mM EDTA, 0.5mM EGTA, 10mM HEPES, and 200mM NaCl), and sonicated three times for 10 seconds at maximum settings in 250 mL lysis buffer (10mM EDTA, 50mM Tris-HCl, 1% SDS, and 0.5% Empigen BB). After 15-minute centrifugation, 10 ML of the supernatant was taken as input and the remainder was diluted 5-fold in immunoprecipitation buffer (2mM EDTA, 100mM NaCl, 20mM Tris-HCl, and 0.5% Triton X-100). This was then subjected to immunoprecipitation overnight with specific antibodies after preclearing with pre-immune IgG, 2mg salmon sperm DNA, and 60mL protein A/G Sepharose bead slurry. Precipitate complexes were serially washed with 300 mL washing buffer I (2mM EDTA, 20mM Tris-HCl, 1% SDS, 01% Triton X-100, and 150mM NaCl), washing buffer II (2mM EDTA, 20mM Tris-HCl, 1% SDS, 01% Triton X-100, and 250mM NaCl), washing buffer III (1mM EDTA, 10mM Tris-HCl, 1% NP40, 1% deoxycholate, and 0.25M LiCl) then twice with 1 mM EDTA and 10mM Tris-HCl. Complexes were removed from the beads through subsequent 15-minute incubations, vortexing, and 5-minute centrifugations with 50 ML of 1% SDS, 0.1 mol/L NaHCO3. Cross-linking was reversed overnight at 65oC and the DNA was purified with QIAquick columns (Qiagen). Anti-BCLAF1 antibody (A300-608A Bethyl Labs) was used for ChIPs
Label
Cy3
Label protocol
1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
Channel 2
Source name
Input from 293T cells transfected with siGFP control siRNA and treated with 1uM Etoposide for 16 hours
293T cells were reverse transfected with GFP or BRCA1 targeted siRNAs using Lipofectamine RNAiMax as per the manufacturers protocol. Following transfection, cells were grown for 72 hours in DMEM before fixing for ChIP.
Extracted molecule
genomic DNA
Extraction protocol
5x10E6 cells were fixed with 1.5% formaldehyde Chromatin was cross- linked using 1.5% formaldehyde for 15 minutes at room temperature. Cells were then washed twice with PBS and collected in 1 mL collection buffer [100mM Tris-HCL (pH 9.4) and 100mM DTT]. The cell suspension was then incubated on ice for 15 minutes. Cells were then lysed sequentially by resuspension and 5-minute centrifugation at 3,000g at 4oC with 1 mL buffer A (10mM EDTA, 0.5mM EGTA, 10mM HEPES, and 0.25% Triton X-100) and 1 mL buffer B (1mM EDTA, 0.5mM EGTA, 10mM HEPES, and 200mM NaCl), and sonicated three times for 10 seconds at maximum settings in 250 mL lysis buffer (10mM EDTA, 50mM Tris-HCl, 1% SDS, and 0.5% Empigen BB). After 15-minute centrifugation, 10 ML of the supernatant was taken as input and the remainder was diluted 5-fold in immunoprecipitation buffer (2mM EDTA, 100mM NaCl, 20mM Tris-HCl, and 0.5% Triton X-100). This was then subjected to immunoprecipitation overnight with specific antibodies after preclearing with pre-immune IgG, 2mg salmon sperm DNA, and 60mL protein A/G Sepharose bead slurry. Precipitate complexes were serially washed with 300 mL washing buffer I (2mM EDTA, 20mM Tris-HCl, 1% SDS, 01% Triton X-100, and 150mM NaCl), washing buffer II (2mM EDTA, 20mM Tris-HCl, 1% SDS, 01% Triton X-100, and 250mM NaCl), washing buffer III (1mM EDTA, 10mM Tris-HCl, 1% NP40, 1% deoxycholate, and 0.25M LiCl) then twice with 1 mM EDTA and 10mM Tris-HCl. Complexes were removed from the beads through subsequent 15-minute incubations, vortexing, and 5-minute centrifugations with 50 ML of 1% SDS, 0.1 mol/L NaHCO3. Cross-linking was reversed overnight at 65oC and the DNA was purified with QIAquick columns (Qiagen). Anti-BCLAF1 antibody (A300-608A Bethyl Labs) was used for ChIPs
Label
Cy5
Label protocol
1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
Hybridization protocol
The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine, 0.1 ug/ul herring sperm DNA and 10 ug of human COT1 DNA (Invitrogen). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html)
