GSM4987140: BC01; Drosophila melanogaster; RNA-Seq
Total RNA was extracted using Trizol reagent (Sigma). Probes complementary to mbl-exon2 were denatured at 85°C for 3 minutes, and placed on ice immediately. Probes mixture was added to total RNA, mixed well and Incubated for 3h at 37°C rotating. Streptavidin C1 beads from NEB (CAT# S1420S) were prepared following manufacturer instructions. 50µl of resuspended beads were added to the sample, and Incubate for 1h at 37°C rotating. Magnetically separated the beads from samples, and then washed 5X in 8X bead volume (800µl) of 1X Hybridization buffer. For elution we added 100µl of elution buffer and then incubated for 10 min at 95°C. Magnetically separated the elutent from beads, and transferred to fresh tube and extract RNA using TRI reagent (Sigma). Samples were treated with DNaseI (NEB). Magnetically separated the elutent from beads, and transferred to fresh tube and extract RNA using TRI reagent (Sigma). Samples were treated with DNaseI (NEB) and performed the nanopore sequencing as instructed in the manual from oxford nanopore technology kit. Libraries preparation was done following Nanopore technology manufacturer instruction for PCR-cDNA Sequencing Kit (SQK-PCS109) Long reads Nanopore technology.