GSM7288815: WT_rep3; Drosophila melanogaster; RNA-Seq
Pupal brains were dissected in PBS and immediately frozen on dry ice. Totally, 30 brains per genotype were collected at 24h APF (After Puparium Formation). Three independent biological replicates per genotype were used for RNA isolation. Total RNA was isolated using RNeasy Plus Universal Kit (QIAGEN, USA) following the manufacturer's instructions including DNase I treatment using RNase-Free DNase Set and on-column protocol (QIAGEN, USA). The isolated total RNAs were aliquoted for both RNA-seq and qRT-PCR. Library preparation and sequencing were performed at Novogene. One microgram of total RNA was used as input for library preparation. Prior to library preparation, total RNA quality was validated for the following parameters:  degradation and contamination using 1% agarose gel electrophoresis, purity by NanoPhotometer® spectrophotometer (IMPLEN, USA), and integrity using Bioanalyzer 2100 and RNA Nano 6000 Kit (Agilent Technologies, USA). Sequencing libraries were generated using NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's instruction. mRNA was purified from total RNA using oligo dT magnetic beads. Purified mRNAs were fragmented by divalent cations. First-strand cDNA synthesis using random hexamer and M-MuLV RT (RNase H-). The dUTP method was used to synthesize the second strand and generate a strand-specific library. After 3' A overhangs were cleaved by exonuclease, DNA fragments were ligated with NEBNext® hairpin loop adaptor to prepare for hybridization. AMPure XP system (Beckman Coulter, USA) was used for 150-200 bp size selection. The size selected and purified products were treated with USER® Enzyme to open up the NEBNext adapter. Then, PCR was performed with Phusion High-Fidelity DNA polymerase using Universal PCR primers and Index (X) prime. PCR products were purified using the AMPure XP system and quality was assessed by Agilent Bioanalyzer 2100. Clustering was done using cBot Cluster System using PE Cluster Kit cBot-HS (Illumina, USA) according to the manufacturer's recommendation. Sequencing was performed on Illumina Novaseq 6000 (Illumina, USA) to generate paired-end reads.