title: RNA-seq of Drosophila melanogaster: 2 weeks 2 replicate male control
design: Double stranded cDNA library was prepared by using NEBNext Ultra Directional RNA Library Prep kit for Illumina following manufacturer’s protocol from 1 mg of total RNA. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and ProtoScript II Reverse Transcriptase. Second strand cDNA synthesis was subsequently performed using Second Strand Synthesis Enzyme Mix. The ds cDNA was isolate from the second-strand reaction mix using Ampure XP beads. The end-repair reaction was used to create blunt ends on the ds cDNA. To avoid the ligation of blunt ends during the adapter ligation reaction, a single ‘A’ nucleotide was added to the 3′ ends of them. The specific RNA Adapter Indexes supplied in the kit were ligated to cDNA fragments. The PCR process (15 cycles) was used to selectively enrich DNA fragments with adapter molecules on both ends and to amplify the amount of DNA in the library, according to the manufacturer’s protocol. Finally, the PCR products were purified (AMPure XP System). The quantity of libraries was determined using the qPCR method by Rotor-Gene 6000 PCR System (Qiagen, USA) according to the manufacturer’s protocol. Primers matched sequences within adapters flanking an Illumina sequencing library. Before starting qPCR, a control template was selected to measure the libraries for quantification. The quality of the libraries was checked on Agilent 2100 Bioanalyzer using a High Sensitivity DNA chip (Agilent Technologies, USA). The final library products were bands at approximately 260 bp. cDNA libraries were normalized to 4nM, pooled together in equal volumes, and sequenced with 75 bp single-end reads on the NextSeq500 System (Illumina, USA). The sequencing data were stored in FASTQ format. At least 20 million reads were obtained for each sample.