title: GSM2224518: TBPH Nipped-B RNAi Replicate 1; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: BG3 cells were grown to a concentration of 5E6 per mL, then crosslinked by addition of 36% formaldehyde directly to the cell culture media to a final concentration of 1%, and incubation for 10 minutes at room temperature on a rocking platform. The reaction was stopped by addition of glycine pH 7.0 to a final concentration of 0.125 M. Cells were washed once with 1x PBS and once each with Wash Buffers A (10 mM HEPES pH 7.6; 10 mM EDTA pH 8.0; 0.5 mM EGTA pH 8.0; 0.25% Triton X100) and B (10 mM HEPES pH 7.6; 200 mM NaCl; 1 mM EDTA pH 8.0; 0.5 mM EGTA pH 8.0; 0.01% Triton X-100) for 10 minutes each. Cells were resuspended in sonication buffer to a concentration of 1x109/3 mL, and sonicated using a BioRuptor Sonicator for 14-20 cycles of 30 s on, 30 s off. Na-lauroylsarcosine was added to 0.5% and incubated for 10 minutes, rotating. Chromatin was centrifuged at 12,000g for 5 minutes at 4°C to pellet insoluble material, and 200 uL chromatin aliquots, each representing ~50x106 cells, were snap frozen on dry ice. For immunoprecipitation, aliquots of chromatin (10 - 20 x 106 BG3 cells) were adjusted to RIPA buffer in a final volume of 500 μL. Adjusted chromatin was pre-cleared for 2 hours with Protein A agarose beads, then incubated with primary antibody overnight at 4°C. Antibody-chromatin complexes were pulled down by incubation with Protein A agarose beads for 4 hours at 4°C, and beads were washed consecutively with RIPA, LiCl buffer, and TE. Samples were treated with RNAse A for 30 minutes at 37°C, then DNA crosslinks were reversed by overnight treatment at 65°C with Proteinase K, and DNA was recovered using Qiagen MinElute columns or phenol/choloroform extraction and ethanol precipitation. Ion Torrent IonXpress barcoded sequencing libraries were constructed using the Ion Xpress Plus Fragment Library Kit (Life Technologies) according to the manufacturer's directions except that size selection was performed using Beckman Agencourt magnetic beads.
attributes.tag: GEO Accession
attributes.value: GSM2224518