GSM1542534: Input_NCI-H2052_rep1; Homo sapiens; ChIP-Seq
Cells were cross-linked in the presence of 1% formaldehyde for 10 min at at room temperature. Crosslinking was stopped by addition of 125mM Glycine for 5 min. Cells were rinsed with 1x PBS and scraped off. Pellets were resuspended in buffer 1 (10 mM Tris (pH 8.0), 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100) and washed once buffer 2 (10 mM Tris (pH 8.0), 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). Then cells were lysed in lysis buffer (50 mM HEPES/KOH (pH 7.5), 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% DOC, 0.1% SDS, protease inhibitors) and sonicated for 15min using a Covaris E210 (TR24-M (500035) and TR12x12 Tubes (520080), Settings: 5% duty cycle, intensity 4). 70ug of chromatin were incubated over night at 4°C with 7ul YAP antibody (Abcam, [EP1674Y] (ab52771) or 20ul TEAD1 antibody (BD Transduction LaboratoriesT (610922)), and for 2h with 25ul preblocked (tRNA, BSA) Dynabeads protein G. Beads were washed twice with  lysis buffer,  once with DOC buffer (10 mM Tris (pH 8.0), 0.25 M LiCl, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA) and once with TE pH8.0, and bound chromatin was eluted in 1% SDS/0.1 M NaHCO3. After RNase A treatment, cross-linking was reversed by overnight incubation at 65 °C followed by proteinase K digestion. DNA was purified using the Minielute PCR purification kit (Qiagen). A sample of the input chromatin was treated in the same way to generate total input DNA. Input and ChIP-samples in duplicate were sequenced. Libraries were prepared using the NEBNext ChIP-seq library reagent set for Illumina (New England BioLabs Inc.)  omitting the size selection step before PCR enrichment.