Oxford long read sequencing of adult transgenic flies
Thirty adult flies were collected and frozen and stored at -80C. Flies were crushed with a tight-fitting pestle in 270 L Homogenization Buffer (HB, 30 mM Tris-HCl pH 8.0, 10 mM EDTA, 0.1 M NaCl, 0.5% Triton X-100). 250 L additional HB was used to wash the pestle (total volume ~520 L). Homogenized flies were centrifuged at 16,000 x g for 5 minutes, before discarding the supernatant. Pellet was resuspended in 520 L HB by gentle inversion, repeating the centrifugation and resuspension. 60 L 20% SDS and 20 L Proteinase K (10 mg/mL) were added followed by incubation at 55C for 1-4 hours, gently finger flicking every 10 minutes. 1 L RNase A (100 mg/mL) was added during the final 30 minutes.  The mixture was centrifuged at 16,000 xg for 5 minutes and the supernatant carefully transferred into a Phase Lock Gel tube (quantBio 2302830). The supernatant was extracted with 1 volume of phenol:chloroform:isoamyl alcohol twice, and once with equal volume of chloroform:isoamyl alcohol and then the aqueous phase transferred to a 2 mL tube using a wide-bore pipette tip. DNA was precipitated with 0.1 volume of 3M sodium acetate and 2 volumes of ice cold 100% ethanol. DNA was pelleted by centrifugation at 10,000 x g for 5 min. The pellet was washed with 1 mL 70% ethanol twice, before letting it air dry at room temperature for 10 minutes. The pelleted DNA was dissolved in 50 L nuclease-free water at 4C overnight, pipetting with a wide-bore 200 L tip if the pellet remained gelatinous. Qubit and spectrophotometer reading were used to assess DNA quality as outlined in the Oxford Nanopore Native barcoding kit (SQK-NBD114.24) using the standard protocol.