GSM6469484: RNAseq_exp1_NELFeKD_DMSO_rep1; Drosophila melanogaster; RNA-Seq
RNA was extracted from the same S2 cell material (with mouse embryonic fibroblasts spiked-in) used for the PRO-seq libraries. For each condition, ~1.9 million S2 cells were used, and total RNA was extracted using TRIzol LS followed by ethanol precipitation and DNase treatment (1hr @ 37˚C with 2U DNase I), followed by a second TRIzol LS extraction and ethanol precipitation. RNA was quantified using Qubit RNA BR assay. 400 ng of total RNA was used for RNA-seq library preparation using the QuantSeq 3' mRNA-Seq Library Prep Kit (FWD) (Lexogen A01172), which uses oligo-dT to prime for first strand synthesis and random priming for second strand synthesis (which is followed by single-ended sequencing in the forward/sense direction of the mRNA). cDNA was prepared using the manufacturer's instructions. The manufacturer's UMI Second Strand Synthesis Module was used to add UMIs for detection of PCR duplicates. Final libraries were PCR amplified (12 cycles) and quantified using the Qubit dsDNA HS assay.