GSM3173047: MTgal4_shSv210_polII; Drosophila melanogaster; ChIP-Seq
Total RNA from ovary was isolated with Ribozol reagent, and rRNA was depleted with Ribo-Zero™ rRNA Removal Kit supplemented with Drosophila rRNA oligonucleotides according to the manufacturer's instruction. Ovaries were fixed using Paraformaldehyde (PFA) at a final concentration of 1% in PBS  for 10min at RT. Samples were quenched by 125mM glycine for 5 min at RT and washed 3 times in PBS. Ovaries were afterwards slightly dounced in Farnham Buffer (5mM HEPES pH8.0; 85mM KCl; 0.5% NP-40/Igepal; NaCl Protease Inhibitor Cocktail; NaF 10mM; Na3VO4 0.2mM) followed by strong douncing in RIPA Buffer (20mM Tris pH7.4; 150mM; 1% NP-40/Igepal; 0.5% Sodium Deoxycholate; 0.1% SDS; Protease Inhibitor Cocktail; NaF 10mM; Na3VO4 0.2mM). Lysates were sonicated using Bioruptor (Diagenode, 20 cycles of 30sec ON, 30sec OFF). Supernatants were pre-cleared for 2h at 4C using Dynabeads Protein G (Invitrogen) beads. 5% of samples were saved for inputs. The rest of the lysates were incubated with indicated antibodies pre-conjugated to protein G dynabeads (Invitrogen) for 2h to overnight at 4C. The beads were washed 5 times at 4C with LiCl IP wash buffer (10mM  Tris pH 7.4;  500mM  LiCL;  1%NP40/Igepal;  1%  Sodium  Deoxycholate) and once with TE. Protein was digested with proteinase K for 3hrs at 55C and reverse-crosslinked by incubation at 65C overnight. DNA was extracted by phenol:chloroform standard protocol. RNA-seq libraries were made using the TruSeq RNA prep kit by Illumina (shW, shSv210-1, shSv210-2) or rthe NEBNext® Ultra™ Directional RNA Library Prep Kit. Libraries were sequenced on the Illumina HiSeq 2000 platform. ChIP-seq library construction was carried out using the NEBNext ChIP-Seq Library Prep Master Mix Set (E6240) with minor modifications. After adaptor ligation and PCR amplification, size selections were done on a 2% agarose gel to select the 200bp-400bp window size window. Libraries were sequenced on the Illumina HiSeq 2000 platform (SR 50bp).
GEO Accession
GSM3173047