Deep whole genome DNA sequencing of 9 Drosophila melanogaster inbred lines
Deep whole genome DNA sequencing of 9 Drosophila melanogaster inbred lines
Adult DGRP and virginizer flies were selected and snap-frozen in liquid nitrogen. We collected 10 adult flies from each of the parental lines used in this study were collected and snap frozen: DGRP-28, DGRP-307, DGRP-399, DGRP-57, DGRP-639, DGRP-712, DGRP-714, DGRP-852 and VGN. To extract the genomic DNA, the flies were crushed with a handheld pestle until powdered, while kept frozen into liquid nitrogen. 500 ml buffer A (Tris-HCl pH 7.5 100 mM, EDTA pH 8.0 100mM, NaCl 100mM, SDS 1%) were added and mixed with the powdered flies. 20 ml of PK proteinase K (PK) 20 mg/ml were added and the samples incubated for 3h at 65C. PK was inactivated by incubation at 95C for 10 min. The samples were then treated with 1 ml RNAseA 10 mg/ml and incubated for 30 min at 37C.  DNA was extracted with 500 ml phenol/chlorophorm/IAA, followed by 500 ul chlorophorm. The aqueous phase was transferred to a new tube and 3 volumes of 100% ethanol were added. The pellet was washed with 1 ml 70% EtOH and air-dryed and resuspended in 10mM Tris-HCl. 2 mg of gDNA per sample were diluted in 100 ml of TE buffer. The gDNA was sonicated with a Bioruptor Pico using 6 cycles of 30s ON and 90s OFF. 50 ml (corresponding to 1 mg of sheared gDNA) were used for library preparation with the NEBNext Ultra II DNA library kit. We enriched for fragments of 300-400bp of length and amplified the library with 3 PCR cycles.