title: GSM1941060: dipchip_msl2_s2_2; Drosophila melanogaster; OTHER
library_construction_protocol: Pellet from 60x106 S2 or Kc cells was resuspended in 1.2 ml of lysis buffer (10mM Tris pH8, 100mM NaCl, 25mM EDTA pH8, 0.5% SDS, 0.15mg/ml of proteinase K) and incubated at 56°C overnight. After addition of 3M sodium acetate, the nucleic acids were subjected to phenol-chloroform extraction and precipitated with equal volume of isopropanol at -20°C for 1 hour. Dried nucleic acids were then resuspended in TE buffer and sonicated with covaris (microTUBEs, Peak Incident Power 175W, Duty Factor 10%, Cycles per Burst 200, 430 seconds) to generate 200bp fragments. After RNAse digestion (0.1 mg/ml, 1h at 37°C), DNA was finally purified the GenElute kit from SIGMA. Synthetic DNA was produced using the Repli-g kit (QIAGEN) with 20ng of gDNA as starting material. Dip-seq experiments were performed as in Gosset at al. 2008 with few modifications. Briefly, 400ng of genomic DNA prepared as specified was incubated with 80 nM of MSL2-FLAG or mutated recombinant protein at 26°C for 30 minutes in 100ul of binding buffer (100mM KCl, 2mM MgCl2, 2mM Tris-HCl pH 7.5, 10% Glycerol, 10uM ZnCl2). For DIP experiments in presence of synthetic DNA, 10pM of the specified synthetic DNA was also added to the reaction. 10% of the reactions was taken as input material and subjected to deep sequencing. DNA-protein complexes were pulled-down using 15ul of FLAG beads slurry (M2, SIGMA) for 15 minutes at room temperature and washed twice with 100ul of binding buffer to eliminate unbound DNA. After proteinase K digestion (0.5mg/ml, 1h at 56°C), DNA was then purified with the GenElute kit from SIGMA and subjected to quantitative PCR and/or deep sequencing. The DIP experiments in presence of synthetic DNA were performed using the deltaRING construct. The Diagenode MicroPlex library kit was used to prepare libraries from 1-2ng of either input or DIP DNA quantified using the Qubit® dsDNA HS Assay kit (Life Technologies Q32851)
identifiers.namespace: GEO
identifiers.id: GSM1941060