GSM6586622: RevIH2i2-gDNA_nanopore_bio rep 1; Drosophila melanogaster; OTHER
Crush 150 frozen flies in 10mL of nuclear isolation buffer (0.35 M sucrose, 0.1 M EDTA, 50 mM Tris-HCl) and homogenise the sample using TissueRuptor II with 2 x 15 second pulses on speed 2. Place 2 layers of 200 μm nylon mesh into a fresh 15 ml Falcon tube. Using a 1 ml wide-bore tip, transfer the homogenised flies through the mesh into the Falcon tube. Wash the nylon mesh with 2 ml of the nuclear isolation buffer. Repeat this wash step one more time. Centrifuge the filtered solution at 3500 x g for 15 minutes at 4 o C. Discard the supernatant. Add 5 ml of Buffer G2 of the QIAGEN Blood and Cell Culture DNA Midi Kit and 95 μl of Proteinase K to the pellet and resuspend by pipetting up and down with a 200 μl wide-bore pipette tip. Incubate at 50 o C for 45 minutes with gentle mixing at 100 rpm. Equilibrate a QIAGEN Genomic-tip 100/G column with 4 ml of Buffer QBT. Pour the lysate through the column. Purify the lysate according to the standard protocol of the QIAGEN kit. Elution is in 150 μl TE buffer. This work has benefited from the facilities and expertise of the high throughput sequencing core facility of I2BC (Centre de Recherche de Gif – http://www.i2bc.paris-saclay.fr/) Instrument: GridION version GXB02022 – 21.02.5; Sequencing kit: SQK-LSK109; Flowcell type: flo-min106 (R9.4.1)