GSM3344624: genomic-DNA-IP_GFP; Drosophila melanogaster; OTHER
Genomic DIP–seq experiments. The pellet from 1 × 108 S2 cells was suspended in 1 ml of lysis buffer (10 mM Tris pH 8, 25 mM EDTA, 100 mM NaCl, 0.1% SDS) and treated with protease K overnight at 55 °C. The DNA is tested with Bradford assay and protein gel to ensure that no proteins remain. DNA is sonicated with Covaris sonicator (power 175 W, duty factor 10%, cycles per burst 200, 250 s) to obtain fragment size average of 200-bp. DNA was purified with the DNA Clean & Concentrator (Zymo).  Proteins were purified using FLAG bead slurry (sigma). The beads were incubated with the extracts for 2 hours followed by 4 washes in HEMGT buffer (25 mM HEPES, pH 7.9, 12.5 mM MgCl2, 150 mM KCl, 10% glycerol). DIP-seq experiment was performed with 1μg of genomic DNA and 1.5 μg of FLAG-NSL3 or FLAG-GFP at 4 °C for 2 hours in 500 μl of DIP buffer (2 mM Tris-HCl pH 7.5, 100 mM KCl, 2 mM MgCl2, 10% glycerol, 10 μM ZnCl2). The FLAG bead was washed six times with 1ml of DIP buffer. Elution was performed with 3X FLAG peptide (100μg/ml, overnight) or digestion with proteinase K (1 h at 56 °C), DNA was purified with the DNA Clean & Concentrator (Zymo). The sequencing was performed using the Illumina HiSeq platform with a 75-bp paired-end kit.