Silencer Negative Control No. 2 siRNA, ThermoFisher Cat#AM4613
Sequenced DNA Library
ChIP-seq was performed using 1x107 siRNA transfected HEK293. Cells were transfected for 1 or 4 days for H3K18ac (814) and H3K27ac (Active Motif) ChIP, 4 days for H3K4me1 (abcam), RAD21 (abcam), and 8 days for YAP (CST D8H1X), TEAD1 (BD Transduction Laboratories), and TEAD4 (Santa Cruz N-G2). For H3K18ac, H3K27ac, H3K4me1 and RAD21 ChIP-seqs cells were cross-linked for 1% formaldehyde for 10 minutes at room temperature on rotator. Formaldehyde crosslinking was quenched with 0.14M glycine for 30 minutes at room temperature on rotator. Cells were washed with PBS and scraped from plates in PBS with Roche protease inhibitor cocktail. Cells were pelleted and lysed in 400uL lysis buffer (1% SDS, 50mM Tris-HCl pH8, 20mM EDTA, Roche complete protease inhibitors) and sonicated at 4°C using the Qsonica Q800R2 at 20% amplitude 10s on 30s off until DNA fragments from sheared chromatin were mostly between the sizes of mostly ~200-600 base pairs. Samples were normalized for equal amounts of DNA as measured by Qubit fluorometer in sonicated, cross-linked chromatin prior to pre-clear and IP. Up to 100uL of sonicated chromatin was diluted in 10X lysis dilution buffer (16.7 mM Tris-HCl, 1.1% Triton X-100, 1.2mM EDTA, 167mM NaCl) and precleared for 1h 4°C with 30uL of protein A dynabeads washed 10X lysis dilution buffer on nutator. IPs were performed O/N at 4°C on nutator with precleared chromatin and 2ug of antibody or 5uL of H3K18ac anti-rabbit sera. 50uL of protein A dynabeads were added for 4h on nutator at 4°C. 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 65°C. 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. YAP, TEAD1, and TEAD4 ChIP-seqs were performed similarly with the following modifications: cells were 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 4°C 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 4°C 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, 2ug of antibody was used, magnetic beads were washed and blocked in 0.5% BSA in PBS. 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.