title: GSM8114854: WCE_OSC_ectopic_ovo_rep2; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: >30 million OSCs were nucleofected with ovo-B-FLAG construct (0.5-0.8 μg per million cells; Nucleofector Kit V, VVCA-1003; NucleofectorTM 2b Device, T-029 program, Lonza) and crosslinked 48 hours later by covering cells in 1% formaldehyde (FA) solution (50mM Hepes-KOH, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 1% FA) for 10 min at room temperature. Cell lysis was performed as described in Schmidt, D. et al. 2009. Sonication was performed with 15-18 cycles of 10 s ON and 60 s OFF (Fisherbrand™ Q705 Sonicator, 418-21 probe) in 3 mL of LB3 buffer (10 mM Tris–HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na–Deoxycholate; 0.5% N-lauroylsarcosine; in 7 ml conical polypropylene tube) to shear DNA into 100-500 bp fragments. Chromatin immunoprecipitation was performed using 5-10 μg of primary mouse monoclonal ANTI-FLAG® M2 antibody (F1804-200UG; Merck) (in 250 μL LB3 with 1% Triton X-100) overnight at 4 °C. Crosslinks were reversed by incubation at 65 °C for 16 h. Proteins and RNA were enzymatically digested. DNA purification was performed using phenol-chloroform extraction and ethanol precipitation. The purified ChIP DNA and 220 ng of whole cell extract DNA were used to prepare ChIP-seq libraries using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (E7645S; with PCR amplifications carried out for 16 cycles) and sequenced in NovaSeq 6000 System with 2 × 150 bp reads on SP flow cell.