title: GSM3381135: ChIP_CLAMP_gst_RNAi_2; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: ChIP‑seq was performed as previously described (Straub et al., 2013) with slight modification. S2 cells (~1x10e8 cells) after RNAi were harvested and chilled on ice. Cells were cross‑linked with 1% formaldehyde for 55 min on ice and the reaction was stopped by adding glycine. Isolated nuclei were resuspended in RIPA (10 mM Tris/HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% (v/v) Triton‑X 100, 0.1%(v/v) SDS, 0.1% (v/v) DOC) at 1x10e8 cells/mL in 1 mL for shearing with Covaris AFA S220 using 12x12 tubes at 100 W peak incident power, 20% duty factor and 200 cycles per burst for 25 min at 5°C to generate 150‑200 bp fragments. 100 µL soluble chromatin was pre‑cleared with 3 µL (6 µL 50% slurry) Protein A:Protein G (1:1) beads (GE Healthcare) and supernatant was directly used for immunoprecipitation by adding antibody to 200 µL chromatin adjusted to 500 µL with RIPA and incubated for 16 h at 4°C. 100 µL supernatant was added to 3 µL (6 µL 50% slurry) Protein A:Protein G beads (1:1) and icubated for 4 h at 4°C. Beads were washed five times with RIPA. For DNA recovery, beads were resuspended in 6.7 bed volumes TE Buffer (10 mM Tris/HCl pH 8.0, 1 mM EDTA), RNA was digested with 50 µg/mL RNaseA (Sigma‑Aldrich) for 30 min at 37°C, and protein was digested with 0.5 µg/mL Proteinase K (Sigma‑Aldrich) supplemented with 0.5% (v/v) SDS for 16 h at 65°C. DNA was purified with 1.8x AMPure XP beads (Beckmann Coulter). Libraries were prepared with NEBNext ChIP‑Seq Library Perp Kit for Illumina (NEB).
identifiers.namespace: GEO
identifiers.id: GSM3381135