GSM9290597: wtOSC_ONT_directRNAseq; Drosophila melanogaster; RNA-Seq
Total RNA was isolated from 90% confluent T75 flasks of OSC cells. Cells were washed three times with PBS, lysed with 10 ml of Trizol, and incubated on a shaker for 30–60 minutes. The lysate was transferred to a 15 ml Falcon tube containing phase lock gel, mixed with 2 ml of chloroform by careful inversion, and centrifuged at 4800 g for 10 minutes at 20°C using a swing-out rotor. The aqueous phase was transferred to a fresh Falcon tube, 7 ml of isopropanol was added, and the mixture was incubated at room temperature for 15 minutes. RNA was then pelleted by centrifugation at 20,000 g for 1 hour at 4°C in a fixed-angle rotor. The supernatant was removed, and the pellet was washed with 75% ethanol. After complete removal of ethanol, the RNA pellet was resuspended in 500 µL of nuclease-free water without pipetting for mixing, and carefully transferred to an Eppendorf tube. RNA concentration was measured using Qubit. mRNA was selected using oligo(dT) Dynabeads. A 200 µL aliquot of Dynabead slurry was washed twice with 2x binding buffer (10 mM Tris HCl pH 7.5, 1.0 M LiCl, and 2 mM EDTA) and resuspended in 100 µL of 2x binding buffer. Total RNA was heat-denatured at 65°C for 5 minutes and snap-cooled on ice. Washed beads were added to the heat-denatured RNA, and the mixture was incubated with overhead rotation for 15 minutes. After incubation, the supernatant was removed, and the beads were washed three times with 500 µL of wash buffer (10 mM Tris HCl pH 7.5, 0.15 M LiCl, and 1 mM EDTA), resuspending the beads each time. mRNA was eluted into 12 µL of nuclease-free water by incubation @ 80°C for 1 minute. mRNA concentration was measured using Qubit. Nanopore direct RNA sequencing libraries were prepared from 10 µL of eluted mRNA following the RNA002 protocol (Oxford Nanopore Technologies). Half of the prepared library was used on a fresh flow cell for sequencing.