GSM6964907: SG_oregon_wt-2; Drosophila melanogaster; RNA-Seq
Total RNA from 25 salivary glands or 25 pairs of wing imaginal discs was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. To construct RNA-seq libraries, we used NEBNext Ultra II RNA Library Prep Kit for Illumina with NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). 1000 ng of RNA was used as a starting material. For imaginal discs, we followed the manufacturer's protocol, but for salivary glands it was modified, because the standard protocol did not allow for complete removal of the ribosomal RNA. To address this issue, mRNA enrichment procedure using oligo-dT beads was repeated twice. Specifically, following the first round of enrichment, mRNA was eluted in 50 ul of TE buffer preheated to 80°С and incubated with beads for 2 minutes (rather than eluted into the First Strand Synthesis Reaction Buffer as in the manufacturer's protocol). The eluate was used for the second round of enrichment on oligo dT beads and processed as described in the protocol. Constructed RNA-seq libraries were sequenced with DNBSeq-G400 platform using 2x150bp paired-end mode (BGI, China). Three technical replicates were run.