HeLa cells were human IFNg-induced for 6 hrs, cross-linked with 1% formaldehyde at room temperature for 10 min, washed twice with ice-cold PBS, collected in 1 ml of PBS (3 x 107 cells per tube) and centrifuged in a bench-top microfuge for 5 min at 5000 r.p.m. Cells were resuspended in 1ml lysis buffer (50mM Tris-Cl pH 8.1, 10mM EDTA, 1% SDS) plus protease inhibitors, incubated on ice for 10 min and sonicated to an average size of 500 bp. Immunoprecipitation (IP) was performed overnight at 4°C with no antibody, anti-STAT1, anti-IRF1, anti-H3ac or anti-H4ac antibody. A aliquot of blocked Staph A cells (Calbiochem, 507862) were added per IP and incubated at RT for 15 min. Precipitates were washed sequentially with 1X dialysis buffer (2 mM EDTA, 50 mM Tris-Cl pH 8.0, 0.2% Sarkosyl) twice, IP wash buffer (100 mM Tris-Cl pH 9.0, 500 mM LiCl, 1% NP40, 1% deoxycholic acid) four times. Samples were extracted with IP elution buffer (50 mM NaHCO3 and 1% SDS), heated at 65°C overnight to reverse cross-links, and DNA fragments purified with QIAEX II gel extraction Kit. LM-PCR was performed using the annealed linker primers oJW102 (5'-GCGGTGACCCGGGAGATCTGAATTC-3') and oJW103 (5'-GAATTCAGATC-3'). Chromatin DNA was blunted using T4 DNA polymerase at 37oC for 45~60 min, and purified using Qiaquick PCR purification kit. Blunted chromatin DNA was ligated to the linker at 16oC over night and purified. The sample was amplified with the following conditions: one cycle at 55oC for 2 min, 72oC for 5 min and 95oC for 2 min; followed by 20 cycles at 95oC for 30 seconds, 55oC for 30 seconds and 72oC for 1 min and one cycle at 72oC for 4 min. DNA was finally purified using Qiaquick.
Label
Cy5
Label protocol
For NimbleGen arrays, ChIP DNA prepared from LM-PCR was directly labeled by Klenow (New England Biolabs) random priming with Cy5 nonamers or Cy3 (ChIP DNA isolated from different antibody immunoprecipitated cells) per manufacturer's protocol.
Channel 2
Source name
Input DNA from HeLa cells without IFNg treatment for 6 hours
HeLa cells were human IFNg-induced for 6 hrs, cross-linked with 1% formaldehyde at room temperature for 10 min, washed twice with ice-cold PBS, collected in 1 ml of PBS (3 x 107 cells per tube) and centrifuged in a bench-top microfuge for 5 min at 5000 r.p.m. Cells were resuspended in 1ml lysis buffer (50mM Tris-Cl pH 8.1, 10mM EDTA, 1% SDS) plus protease inhibitors, incubated on ice for 10 min and sonicated to an average size of 500 bp. Immunoprecipitation (IP) was performed overnight at 4°C with no antibody, anti-STAT1, anti-IRF1, anti-H3ac or anti-H4ac antibody. A aliquot of blocked Staph A cells (Calbiochem, 507862) were added per IP and incubated at RT for 15 min. Precipitates were washed sequentially with 1X dialysis buffer (2 mM EDTA, 50 mM Tris-Cl pH 8.0, 0.2% Sarkosyl) twice, IP wash buffer (100 mM Tris-Cl pH 9.0, 500 mM LiCl, 1% NP40, 1% deoxycholic acid) four times. Samples were extracted with IP elution buffer (50 mM NaHCO3 and 1% SDS), heated at 65°C overnight to reverse cross-links, and DNA fragments purified with QIAEX II gel extraction Kit. LM-PCR was performed using the annealed linker primers oJW102 (5'-GCGGTGACCCGGGAGATCTGAATTC-3') and oJW103 (5'-GAATTCAGATC-3'). Chromatin DNA was blunted using T4 DNA polymerase at 37oC for 45~60 min, and purified using Qiaquick PCR purification kit. Blunted chromatin DNA was ligated to the linker at 16oC over night and purified. The sample was amplified with the following conditions: one cycle at 55oC for 2 min, 72oC for 5 min and 95oC for 2 min; followed by 20 cycles at 95oC for 30 seconds, 55oC for 30 seconds and 72oC for 1 min and one cycle at 72oC for 4 min. DNA was finally purified using Qiaquick.
Label
Cy3
Label protocol
For NimbleGen arrays, ChIP DNA prepared from LM-PCR was directly labeled by Klenow (New England Biolabs) random priming with Cy5 nonamers or Cy3 (ChIP DNA isolated from different antibody immunoprecipitated cells) per manufacturer's protocol.
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
Scan protocol
Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol
Description
Analysis of H3ac, H4ac, STAT1 and IRF1 binding in IFNg treated and untreated HeLa cells for 6 hours was done using 50mer oligonucleotide probes at 30bp intervals tiling over non-repetitive 16MB selected genes (HG17).
