title: GSM2828236: ChIP-seq for Input in HAP1 cell line treated with DMSO, replicate 2; Homo sapiens; ChIP-Seq
library_construction_protocol: For each immunoprecipitation 30 million nuclei were isolated. Cells were fixed with 1% paraformaldehyde for 10 min at room temperature. The fixation was stopped by addition of glycine. The collected pellet was sonicated using a Covaris S220 sonicator for 43 minutes to fragment the DNA to 200-700 bp. For immunoprecipitation, the sonicated chromatin was added to antibody-conjugated Protein A or G beads (Life Technologies) and incubated rotating at 4°C overnight. Used antibodies were: anti-MTHFD1 (C3, Santa Cruz), anti-BRD4 (A301‐985A, Bethyl Labs), anti-H3K27Ac (ab4729, Abcam) and mouse IgG (sc-2025, Santa Cruz). Tagmentation was performed by resuspending the magnetic beads in 100 μl tagmentation reaction mix (10 mM Tris pH 8.0, 5 mM MgCl2, 10 % v/v dimethylformamide) containing 2 μl Tagment DNA Enzyme from the Nextera DNA Sample Prep Kit (Illumina) followed by an incubation for 3 min at 37°C. Subsequently, formaldehyde crosslinks were reverted by incubation in elution buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA and 10 mM Tris-HCl pH 8.0) containing 2 µl of Proteinase K for 1 h at 55°C, and then at 65°C overnight. The DNA was purified using the QIAquick PCR Purification kit (Qiagen). The enrichment of the libraries was performed in a 50 µl-reaction using Kapa HiFi HotStart ReadyMix (Kapa Biosystems) and 0.75 μM primers. Each DNA library was purified and size selected for a fragment length of 200-400 bp using SPRI AMPure XP beads (Beckman Coulter).
identifiers.namespace: GEO
identifiers.id: GSM2828236