GSM4910627: B_Rpb3.dom-kd.st5.rep1; Drosophila melanogaster; ChIP-Seq
ChIP-seq experiments were performed as in Zenk et al., 2017. Briefly, nuclei were extracted in lysis buffer (140 mM NaCl, 15 mM HEPES [pH 7.6], 1 mM EDTA, 0.5 mM EGTA, 1 % TritonX100, 0.5 mM DTT, 0.1 % Sodium Deoxycholate, 10 mM Sodium Butyrate, 1X Protease Inhibitors) and subsequent ultrasound treatment (Covaris E220, 45 sec, peak power 75, duty factor 10, cycles burst 200). Fixed chromatin was then sheared using Covaris E220 (900 sec, peak power 140, duty factor 5, cycle burst 200) and precleared overnight. Each sample was incubated with the relevant antibody and concentration for at least 4 hrs at 4°C. Samples were then washed, eluted and decrosslinked overnight by incubation at 65°C. Next, samples were treated with RNaseA(50mg/mL final concentration) for 30min at 37°C and ProteinaseK (200mg/mL, final) for 3 hrs at 56°C. Libraries were prepared according to manufacturer's instructions using the NEBNext Ultra II DNA Library Prep Kit for Illumina. Libraries were quality controlled by capillary electrophoresis on the Fragment Analyzer system (Advanced Analytical).