GSM6579011: Vehicle Control A375 JUNB 11.18.20; Homo sapiens; ChIP-Seq
A375 cells were fixed directly in 15cm plates with 11% formaldehyde and collected by scraping. Approximately 100,000,000 cells were used per condition. Cells were lysed using lysis buffers with protease inhibitors (Roche #05056489001) and sonicated such that fragmented chromatin was 200-300bp long. Optimal chromatin length was confirmed by gel electrophoresis. Prior to antibody addition, 50µL chromatin was collected for input sample. The remaining sonicated chromatin was incubated overnight at 4°C with 10µg antibody attached to Dynabeads (Invitrogen #10004D). Samples (including inputs) were washed with wash buffers and eluted for 6 hours at 70°C, treated with RNaseA (Sigma #R4642) and ProteinaseK (Life Technologies #AM2546), and purified using Zymo ChIP DNA Concentrator kit (Genesee Scientific #11-379C). Libraries were end repaired (VWR #ER81050), polyA tailed (Invitrogen #18252-015, NEB #M0212L), adaptor ligated (NEB #E7335), size selected using Ampure XP beads (Beckman Coulter #A63881, Life Technologies #12027), and PCR amplified (NEB #M0531).