GSM5050835: MSl2mel DIP1; Drosophila virilis; ChIP-Seq
Pellet from 6 × 107 S2 or virilis cells was suspended in 1.2 ml of lysis buffer (10 mM Tris pH 8, 100 mM NaCl, 25 mM EDTA pH 8, 0.5% SDS, 0.15 mg/ml of proteinase K) and incubated at 56°C overnight. After addition of sodium acetate to a final concentration of 0.3 M, the nucleic acids were extracted with phenol–chloroform and precipitated with an equal volume of isopropanol at −20°C for 1 h. Precipitated nucleic acids were centrifuged and washed with 70% ethanol. Dried pellets were resuspended in TE buffer and sonicated with Covaris AFA S220 (microTUBEs, Peak Incident Power 175 W, Duty Factor 10%, Cycles per Burst 200, 430 s) to generate 200 bp fragments. After RNAse digestion (0.1 mg/ml, 1 h at 37°C), DNA was purified with the GenElute kit (Sigma-Aldrich). DNA immunoprecipitation (DIP) experiments were performed as in (Villa et al. 2016) with few modifications. Briefly, 400 ng of genomic DNA (gDNA) was incubated with 80nM of recombinant proteins at 26°C for 30 min in 100 μl of binding buffer (100 mM KCl, 2 mM MgCl2, 2 mM Tris–HCl pH 7.5, 10% Glycerol, 10 μM ZnCl2). Ten percent of the reaction was taken as input material. DNA–protein complexes were immunoprecipitated using 30 μl of anti-FLAG M2 beads (SIGMA) and washed twice with 100 μl of binding buffer to eliminate unbound DNA. After proteinase K digestion (0.5 mg/ml, 1 h at 56°C), DNA was purified with AMPure beads (Beckman Coulter). Libraries for sequencing were prepared using NEBNext Ultra II DNA library prep kit for Illumina (New England Biolabs, E7465).