GSM3381164: DIP_Input_2; Drosophila melanogaster; OTHER
For genomic DNA (gDNA) extraction, 5x10e7 S2 cells were harvested, washed in PBS and gDNA was extracted with NucleoSpin Tissue kit (Macherey‑Nagel). Remaining RNA contaminants in eluted gDNA were digested with 0.1 mg/mL RNaseA (Sigma‑Aldrich) for 1 h at 37°C. Subsequently, gDNA was sonicated with Covaris AFA S220 using microTUBEs at 175 W peak incident power, 10% duty factor and 200 cycles per burst for 430 s at 5°C to generate ~150‑200 bp fragments, as described in (Villa et al., 2016). Sheared gDNA was purified with MinElute kit (QIAGEN). DIP‑seq was performed accordingly to (Villa et al., 2016), with modifications for combined DIP of two proteins. Sheared gDNA was diluted to 4 mg/mL in Binding Buffer (2 mM Tris/HCl pH 7.5, 100 mM KCl, 2 mM MgCl2, 10% (v/v) Glycerol, 10 µM ZnCl2). Per reaction, 100 nM recombinant MSL2 and/or 50 nM recombinant CLAMP was added to 100 µL diluted gDNA and incubated for 30 min at 26°C. For immunoprecipitation, the reaction was added to 7.5 µL (15 µL 50% slurry) Protein G beads (GE Healthcare) pre‑coupled with antibodies and incubated for 15 min at RT. Beads were washed twice in 100 µL Binding Buffer and resuspended in 50 µL Binding Buffer. Protein was digested by adding Proteinase K at 0.5 mg/mL and incubated for 1 h at 56°C and DNA was purified with 1.8x AMPure XP beads (Beckmann Coulter). Libraries were prepared with MicroPlex Library Preparation Kit v2 (Diagenode).
GEO
GSM3381164
GEO Accession
GSM3381164