GSM8654118: rRNA depleted S2 cells sample; Drosophila melanogaster; RNA-Seq
5-day old adult Drosophila adult brains (N=15) of desired genotypes were dissected in 1X PBS and RNA was extracted using TRIzol method (Thermo Fisher Scientific) following the manufacturer's protocol. Quality of RNA was assessed using Agilent Tapestation 4200 before proceeding for Library preparation and sequencing. For Poly(A)-enrichment of mRNA, Illumina libraries were prepared using NEBNext Ultra II Directional RNA Library Prep kit (E7765L) and sequenced with Illumina NovaSeq 6000 system.rRNA-depletion was performed as recommended by NEBNext RNA Depletion Core Reagent protocol. Briefly, the RNA integrity was confirmed on an Agilent Tapestation 4200 and samples with at least 7.0 RIN values were used for experiments. The total RNA (500-1000 ng), 2 μL of the 2μM probe pool, 2 μL of the 2μM NEBNext probe hybridisation buffer and nuclease free water were mixed to a total volume of 15 μL. This mixture was then incubated at 95°C for 2 min, a touchdown from 95°C to 22°C (0.1°C/sec) and a 5-min hold at 22°C.  We then added 2 μL NEBNext RNase H Reaction Buffer, 2 μL NEBNext Thermostable RNase H and 1 μL nuclease free water and incubated for 30 min at 50°C. Then RNase H treated sample was digested with DNase I and purified using NEBNext RNA Sample Purification Beads as recommended by NEBNext RNA Depletion Core Reagent protocol. The concentrations of purified RNA samples were measured using the Qubit HS RNA Assay. The purified rRNA depleted samples were taken for library preparation using NEBNext Ultra™ II Directional RNA Library Prep with Sample Purification Beads (Catalogue no-E7765L) as per the manufacturer's protocol. The libraries were sequenced on Illumina NovaSeq 6000 platform with 2x100bp read length. Similarly, rRNA-depletion was performed as recommended by QIAseq FastSelect – rRNA Fly Kits protocol (Cat. No. / ID:  333262) using total RNA from Drosphila S2 cells. The rRNA-depleted samples were processed using similar protocol as described above.