GSM2816745: DCAF7b_IRF3_INP; Homo sapiens; ChIP-Seq
1x10^7 low-passage HBTEC were mock-infected or infected with indicated Ad vector 3 days after reaching confluence. 24h p.i. cells double crosslinked with 4mM DSG in PBS for 30min then 1% formaldehyde for 10 min, crosslinking was quenched in 500mM Tris pH7.9 for 20min and cell pellets were lysed in 1mL lysis buffer 1 (50mM HEPES-KOH, pH 7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, Roche complete protease inhibitors) for 10min on ice. Lysate was pelleted at 3000 rpm 5min 4C then resuspended in 1mL lysis buffer 2 (10mM Tris-HCl, pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA, Roche complete protease inhibitors) and placed on nutator 10min 4C and pelleted as before, then resuspended in 125uL of lysis buffer 3 (10mM Tris-HCl, pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, Roche complete protease inhibitors) and sonicated at 4C using the Qsonica Q800R2 at 20% amplitude 10s on 30s off until DNA fragments from sheared chromatin were mostly between the sizes of 200-600 base pairs. 100uL of sonicated chromatin was diluted in 4X lysis dilution buffer (16.7 mM Tris-HCl, 1% Triton X-100, 1.2mM EDTA, 167mM NaCl, 0.1% SDS) and precleared for 1h 4C with 30uL of protein A dynabeads washed and blocked in 0.5% BSA in PBS on nutator. IPs were performed O/N at 4C on nutator with precleared chromatin and 5ug of anti-IRF3 (Cat#sc-9082). 50uL of protein A dynabeads were added for 4h on nutator at 4C. Bead-immunocomplexes were washed for 5min 2X with each of the following buffers in order: wash buffer A (50mM Hepes pH 7.9, 0.1% SDS, 1% Triton X-100, 0.1% Deoxycholate, 1mM EDTA, 140mM NaCl), wash buffer B (50mM Hepes pH 7.9, 0.1% SDS, 1% Triton X-100, 0.1% Deoxycholate, 1mM EDTA, 500mM NaCl), LiCl buffer (20mM Tris-HCl pH8, 0.5% NP-40, 0.5% Deoxycholate, 1mM EDTA, 250mM LiCl), TE (50mM Tris-HCl pH8, 1mM EDTA). Elution was performed in 150uL of elution buffer (50mM Tris HCl pH8, 1mM EDTA, 1% SDS) then ChIP samples and inputs (10uL of precleared chromatin lysis plus 140uL elution buffer) were reverse crosslinked O/N at 65C. Samples were RNase A treated for 1h at 37¡C and DNA was purified and extracted with phenol/chloroform and ethanol precipitated. DNA pellets were resuspended in 12uL of TE and measured using Qubit fluorometer. ChIP sequencing libraries were constructed from 1 ng of immunoprecipitated and input DNA using the KAPA Hyper Prep Kit from KAPA Biosystems and NEXTflex ChIP-Seq barcodes purchased from Bioo Scientific.
GEO Accession
GSM2816745