alias: GSM1920194
title: GSM1920194: X1_input; Schmidtea mediterranea; ChIP-Seq
library_construction_protocol: Chromatin-immunoprecipitation was performed as previously described (Lee et al., 2006) with some modifications. Briefly, worms were snap-frozen in liquid nitrogen, homogenized with a clean razor, resuspended in freshly cracked 4% PFA (EMS)/calcium- and magnesium-free buffer with 1% BSA (CMFB) and rocked for 10 minutes at room temperature to cross-link and dissociate. Samples were then quenched with 0.125M glycine, filtered with 100μm cell strainers (Corning), pelleted by centrifugation at 700*g, and washed twice with ice-cold 0.4XPBS + 10% FBS + 1X Roche Complete protease inhibitors. FACS-isolated planarian cells and Drosophila S2 cells were cross-linked in suspension in collection buffer + 10% FBS or Schneider’s media + 10% FBS, respectively. Pellets were then either snap-frozen or resuspended in lysis buffer (1% SDS, 50mM Tris pH 8, 10mM EDTA) and incubated on ice for 10 min. Samples were then sheared for 12 min in the S220 Focused Ultrasonicator (Covaris) in milliTubes (Covaris) with either the AFA fiber or 100μl glass beads (for volumes < 1ml). Samples were then cleared by centrifugation (10K*g, 10 min) and used for immunoprecipitations. H3K4me3 antibody (Millipore 07-473) was used at 0.5-1μl antibody/μg chromatin (as estimated by Qubit measurement of dsDNA in IP inputs) and incubated 4hrs - overnight at 4°C. Antibody-chromatin complexes were then captured with a mix of ProteinA/G magnetic beads (Life Technologies; 2hrs, 4°C), washed, eluted and incubated overnight at 65°C to reverse cross-links. Samples were then digested with RNase and ProteinaseK, and DNA was isolated by column purification (Qiagen). DNaseq libraries were made using an optimized protocol with reagents from the Illumina TruSeq RNA kit and data was collected for ChIP Seq analysis on an Illumina HiSeq 2500.
identifiers.namespace: GEO
identifiers.id: GSM1920194