title: RNA-Seq of Drosophila melanogaster: pupal eye
design: Following initial QC steps mRNA was isolated and libraries were generated using the Swift Biosciences Rapid RNA kit according to the manufacturers protocol. Briefly, mature mRNA was enriched for via pull down with beads coated with oligo-dT homopolymers using the NEBNext Poly(A) mRNA Magnetic Isolation Kit. The mRNA molecules were then chemically fragmented and the first strand of cDNA was generated using random primers incorporating a truncated i5 adapter sequence. Following a bead-based cleanup the 3 end of the single stranded cDNA was ligated to a truncated i7 adapter. Libraries were then indexed using Swift Biosciences Unique Dual Indexing primers. Final libraries for each sample were assayed on the Agilent Tapestation for appropriate size and quantity. These libraries were then pooled in equimolar amounts as ascertained via fluorometric analyses. Final pools were absolutely quantified using qPCR on a Roche LightCycler 480 instrument with NEB Illumina Library Quantification reagents. Sequencing of the libraries was performed on an Illumina NovaSeq 6000 with PE150 reads.