GSM4879171: w1118_whole_fly_wgs; Drosophila melanogaster; OTHER
Whole Genome Sequencing (WGS): 100 flies were collected in a 1.5ml tube and frozen at -80°C for at least 1h. High molecular weight genomic DNA (HMW gDNA) was isolated using the Blood and Cell Culture DNA Mini kit (Qiagen). Flies were homogenised using a rotating pestle on ice for 1min. 700µl G2 and 50µl Proteinase K (800U/ml, NEB) was added to each tube and incubated at 50°C for 2h with occasional tube inversions. Tubes were spun at 5,000g for 10min at 4°C and supernatant transferred to new tube avoiding debris. A Qiagen Genomic-tip 20/G was equilibrated with 1ml QBT buffer and allowed to empty by gravity flow. The supernatant containing digested proteins and genomic DNA was added to the column and allowed to flow through. The column was washed 3 times with 1ml QC buffer. Elution was carried out with 1ml QF elution buffer and repeated once. Flow through was transferred to two new tubes (1ml each) and 700µl isopropanol added. Tubes were inverted 10 times and centrifuged at full speed for 15min at 4°C. The pellet was washed with 70% ethanol twice and air-dried for 5min. 25µl RNase-free water was added and DNA resuspended by flicking tube gently several times while incubating at 37°C for 2h. DNA was stored at 4°C. DNA was sheered using a Covaris S220 (Covaris). 3µg of genomic DNA was diluted in RNase-Free water and transferred to a AFA Fiber Crimp-Cap (PN520052, Covaris) microtube. Sonication was carried out with the following settings: Peak incident Power (W) 105, Duty Factor 5%, Cycles per Burst 200, Treatment time 80sec. This resulted in sheared DNA fragments peaking at 500bp. DNA was recovered using the QIAquick PCR Purification Kit (Qiagen). NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® Whole genome sequencing