GSM3860939: MCF7 DMSO; Homo sapiens; ChIP-Seq
For ChIP, Cells were washed in phosphate-buffered saline (PBS) and crosslinked with 1% Formaldehyde for 10 minutes (H3K27Ac) or crosslinked with two agents starting with 2 mM DSG (Pierce) for 45 min at RT, followed by 1 ml 1% Formaldehyde for 10 min (YAP and TEAD4). Crosslinked Cell lines were quenched with 0.125 M glycine for 5 min at room temperature. After quenching, the material was resuspended in 1% SDS (50 mM Tris-HCl pH8, 10 mM EDTA) and sonicated for 5 minutes with a Covaris E220 instrument, 5% duty cycle, 140 Peak Incident Power, 200 Cycles per burst, in 1ml AFA Fiber milliTUBEs. Soluble chromatin was immunoprecipitated with 10 μg of H3K27ac antibody, 7 μg of YAP antibody, 1.5 μg of SLUG antibody, or 1.5 μg of TEAD antibody. 5 µg of chromatin was used for H3K27Ac ChIP, and 40 µg for YAP, TEAD4 and SLUG ChIPs. For ATAC-seq, 50000 cells / sample were resuspended in 1 ml of cold ATAC-seq resuspension buffer (RSB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, and 3 mM MgCl2 in water). Cells were centrifuged at max speed for 5 min in a pre-chilled (4 C) fixed-angle centrifuge. After centrifugation supernatant was carefully aspirated.  Cell pellets were then resuspended in 50 μl of ATAC-seq RSB containing 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin by pipetting up and down three times. This cell lysis reaction was incubated on ice for 3 min. After lysis, 1 ml of ATAC-seq RSB containing 0.1% Tween-20 (without NP40 or digitonin) was added, and the tubes were inverted to mix. Nuclei were then centrifuged for 5 min at max speed in a pre-chilled (4 C) fixed-angle centrifuge. Supernatant was removed and nuclei were resuspended in 50 μl of transposition mix (Corces et al., 2017): 2.5 μl transposase (100 nM final), 16.5 μl PBS, 0.5 μl 1% digitonin, 0.5 μl 10% Tween-20, and 5 μl water) by pipetting up and down six times. Transposition reactions were incubated at 37 C for 30 min in a thermomixer with shaking at 1,000 r.p.m. Reactions were cleaned up with Qiagen columns. ChIP-seq libraries were constructed using Accel-NGS 2S DNA library kit from Swift Biosciences. ATAC-seq libraries were amplified as described previously (Buenrostro et al., 2015).