title: GSM3573311: RNA-seq in HAP1 cell line with MTHFD1 knockout, clone D3, replicate 2; Homo sapiens; RNA-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). RNA extraction was performed with Qiagen RNeasy Mini Kit (Cat No. 74106) according to the manufacturer's instructions. 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). Total RNA was quantified using Qubit 2.0 Fluorometric Quantitation system (Life Technologies) and the RNA integrity number (RIN) was determined using Experion Automated Electrophoresis System (Bio-Rad). RNA-seq libraries were prepared with TruSeq Stranded mRNA LT sample preparation kit (Illumina) using Sciclone and Zephyr liquid handling robotics (PerkinElmer). Library amount was quantified using Qubit 2.0 Fluorometric Quantitation system (Life Technologies) and the size distribution was assessed using Experion Automated Electrophoresis System (Bio-Rad). For sequencing libraries were pooled, diluted and sequenced on Illumina HiSeq 3000 using 50 bp single-read chemistry.