title: GSM3732272: ChIPseq.s2.h3k9me2_jil2_3; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: For ChIP-seq on MNase-digested chromatin in combination with mild sonication, S2 cells (~3 * 10^8 cells) after RNAi treatment were cross‑linked with 1% formaldehyde for 8 min at room temperature and the reaction was stopped by adding glycine. 5% 79f7Dv3 cells, processed as described for S2 cells without RNAi treatment, relative to S2 cells were added. 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) supplemented with PI and 2 mM CaCl2 at 7 * 10^7 cells/mL and digested in 1 mL aliquots by adding 0.6 U MNase (Sigma Aldrich), resuspended in EX50 at 0.6 U/µL (Bonte and Becker, 1999), and incubating at 37°C for 35 min with slight agitation. The reaction was stopped by adding 10 mM EGTA and placing on ice. Digested chromatin was sheared with Covaris AFA S220 using 12x12 tubes at 50 W peak incident power, 20% duty factor and 200 cycles per burst for 8 min at 5°C. Subsequent steps were performed as described in (Albig et al., 2019). In brief, 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 300 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 incubated 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).
attributes.tag: GEO Accession
attributes.value: GSM3732272