title: GSM9385119: YAP_DMSO_treated_parental_MSTO_cells_rep2; Homo sapiens; ChIP-Seq
library_name: GSM9385119
library_construction_protocol: NCI-H226 and MSTO-211 mesothelioma cells (10 x 106) were double crosslinked with 50mM DSG (disuccinimidyl glutarate, #C1104 - ProteoChem) for 30-minutes followed by 10-minutes of 1% formaldehyde. Formaldehyde was quenched by the addition of glycine. Nuclei were isolated with ChIP lysis buffer (1% Triton x-100, 0.1% SDS, 150 mM NaCl, 1mM EDTA, and 20 mM Tris, pH 8.0). Nuclei were sheared with Covaris sonicator using the following setup: Fill level ? 10, Duty Cycle ? 15, PIP ? 350, Cycles/Burst ? 200, Time ? 8-minutes). Sheared chromatin was immunoprecipitated overnight with the following antibodies: FOSL1 (PA5-66880 ? Invitrogen), YAP (14074S ? Cell Signaling), KLF4 (AF3158 ? R&D Systems). Antibody chromatin complexes were pulled down with Protein A magnetic beads and washed twice in IP wash buffer I. (1% Triton, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8.0, and 0.1% NaDOC), twice in IP wash buffer II. (1% Triton, 0.1% SDS, 500 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8.0, and 0.1% NaDOC), twice in IP wash buffer III. (0.25 M LiCl, 0.5% NP-40, 1mM EDTA, 20 mM Tris, pH 8.0, 0.5% NaDOC) and once in TE buffer (10 mM EDTA and 200 mM Tris, pH 8.0). DNA was eluted from the beads by vigorous shaking for 20-minutes in elution buffer (100mM NaHCO3, 1% SDS). DNA was de-crosslinked overnight at 65C and purified with the MinElute PCR purification kit (Qiagen). DNA was quantified by Qubit and equal amounts (2ng) of DNA was used for sequencing library construction with the Ovation Ultralow Library System V2 (Tecan) using 15 PCR cycles according to the manufacturer?s recommendations. Libraries were sequenced with Illumina Nextseq 2000, using paired-end 50bp read configuration.