GSM5322476: M46018_1; Drosophila melanogaster; RNA-Seq
RNA integrity was assessed with the RNA nano 6000 assay kit for the Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). Briefly, mRNA was purified from total RNA with poly T oligo-attached magnetic beads. Fragmentation was conducted at high temperature with divalent cations in 5× first-strand synthesis reaction buffer. To select cDNA fragments 370–420 bp long, library fragments were purified in an AMPure XP system (Beckman Coulter, Beverly, MA, USA). PCR was performed with Phusion high-fidelity DNA polymerase, universal PCR primers, and index (X) primer. The PCR products were purified in the AMPure XP system (Beckman Coulter, Beverly, MA, USA) and library quality was assessed in the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). Index-coded samples were clustered on a cBot cluster generation system using a TruSeq PE cluster kit v3-cBot-HS (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. The reference genome index was built and paired-end clean reads were aligned to the reference genome with Hisat2 v2.0.5. FeatureCounts v. 1.5.0-p3 enumerated the reads mapped to each gene. Differential expression analysis was performed in the DESeq2 package of R v. 1.20.0 (R Core Team, Vienna, Austria). RNA libraries were prepared for sequencing using standard Illumina protocols