title: GSM5701410: MMD4m9_3LW rep 1; Drosophila melanogaster; OTHER
library_construction_protocol: Salivary glands from female wandering third-instar larvae were isolated and flash-frozen in liquid N2 until all samples were collected. Genomic DNA for sequencing was prepared from 25 L3 salivary gland pairs or 10 mg embryos (0-6 h AED) using DNeasy Blood and Tissue kit (Qiagen). Each sample was prepared in triplicate. The tissues were soaked in 180 µl buffer ATL + 20 µl proteinase K (15 mg/ml) and lysed for 2-3 h at 55°C. The reactions were cooled to room temperature, supplemented with 4 µl RNase A, ~40 mg/ml (Sigma Aldrich), and RNA was digested for 10 min at room temperature. The genomic DNA was fragmented with 0.002 units DNase I (Thermo Fisher) in 100-µl reactions containing 10 mM Tris-HCl, pH 7.5, 10 mM MnCl2, 0.1 mM CaCl2, 0.1 mg/ml RNase A and 0.2 mg/ml nuclease-free BSA (1x reaction buffer) for 15 min at 37°C. (DNAse I dilutions were prepared using 1x reaction buffer.) Reactions were stopped by adding 5 µl 0.5 M EDTA, and DNase I was inactivated for 20 min at 65°C. The fragmented DNA was purified on QiaQuick columns using PCR purification kit (Qiagen) and eluted in 40 µl 10 mM Tris-HCl, pH 8.0. The size distribution of DNA fragments (200-600 bp, average ~400 bp) was confirmed and DNA concentration was measured on 2100 BioAnalyzer (Agilent). Libraries were prepared from 20 ng of fragmented genomic DNA with the ThruPLEX DNA-seq kit using SMARTer® DNA Unique Dual Indexes (TakaraBio)