GSM8889015: Sov-KD_2_RNA_rep2; Drosophila melanogaster; RNA-Seq
For RNA-seq, total RNAs were isolated by RNeasy kit (Qiagen). Poly(A)+ RNAs were obtained using oligo-dT beads. For ChIP experiments targeting RNA polymerase II, OSC were fixed in 0.3% formaldehyde (methanol-free, Thermo Fisher) for 10 min at room temperature and quenched with a final concentration of 125 mM glycine for 2 min. After washing twice with PBS (containing 125 mM glycine), nuclei were isolated using 200 µl Low salt lysis buffer (10 mM HEPES-KOH pH 7.4, 10 mM NaCl, 1.5 mM MgCl2, 1% Nonidet P-40) supplemented with cOmplete (Protease inhibitor cocktail, Roche) and PhosSTOP (Phosphatase inhibitor cocktail, Roche). The isolated nuclei were resuspended in 200 µl Sonication buffer (50 mM Tris-HCl pH 7.9, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate) supplemented with cOmplete and PhosSTOP, and sonicated using a probe-sonicator (BRANSON, SFX150) at 30% amplitude, with 2 sec pulses and 3 sec intervals, for a total of 2 min, in an ice-cold water bath. After centrifugation, the supernatant was passed through a 0.22-μm syringe filter (Membrane Solutions) and used for immunoprecipitation.To elucidate Pol II occupancy on the reporter gene, we utilized an anti-Pol II ser5-P antibody (Cosmobio, CMA603).The chromatin lysate was incubated with 1 µg of anti-Pol II ser5-P antibody at 4 °C for 3 h with rotation. The antibody was captured using Dynabeads Protein G.The captured chromatin was extracted in 100 µl of Elution buffer (50 mM Tris-HCl pH 7.9, 10 mM EDTA, and 1% SDS) at 65 °C for 10 min. The supernatant was supplemented with 4 µl of 5 M NaCl, and cross-link were reversed at 65 °C overnight in a Thermomixer at 1400 rpm (Eppendorf). DNA was purified using a spin column and eluted in Milli-Q water. For RNA-seq, Poly(A)+ RNA was isolated from 2 µg total RNA using Dynabeads Oligo (dT)25 (Thermo Fisher), followed by heat-fragmentation at 94ºC for 6 min. cDNA was synthesized from the fragmented RNA by random hexamers and SuperScript III (Thermo Fisher), and then purified by AMPure XP (Beckman). Second strand DNA was synthesized by DNA polymerase I (NEB) in the presence of 1 mM dUTP at 16ºC for 2.5 h, and then purified by AMPure XP. Library preparation was carried out using the NEBNext Ultra II DNA Library Prep kit for illumina (NEB), according to the manufacturer's instructions. For ChIP-seq, ChIPed DNAs were used for library preparation by NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) following the manufacture's protocol.