Hiseq 2000 2500 sequencer

A total of 12 RNA samples were collected from different developmental stages and tissues (Supplementary Table S9). The sample sources included adult flies, embryos (eggs), larvae, pupae as well separate tissues including heads, legs, ovipositor, testes, sex organs, and thorax. Total RNA was extracted using the Trizol method and quality-checked as previously described [28 (link)]. The TruSeq stranded library preparation protocol (Illumina) was followed by 100 bp paired-end sequencing with Hiseq 2000/2500 sequencers (Illumina).

Whole-exome sequencing (WES) was performed using the HiSeq 2000/2500 platform within the Core Facility Management Center of the Korea Research Institute of Bioscience and Biotechnology (KRIBB). The sequencing libraries were prepared from primary DNA extracted from the leukocytes of blood samples using the TruSeq library preparation kit (Illumina, San Diego, CA, USA) following the manufacturer’s protocol. The Nextera Rapid Capture Exome kit (Illumina) was used to selectively amplify the coding regions of the genome according to the manufacturer’s protocol. The captured libraries were sequenced using a HiSeq 2000/2500 sequencer (Illumina), and 2 × 100 bp were utilized for paired-end sequencing according to the manufacturer’s recommendations. Image analysis and base calling were performed using the Illumina pipeline. The sequencing data were mapped to the human reference genome (GRCh37, UCSC hg19) using the Burrows-Wheeler Aligner software. PCR duplicates were removed using Picard software. The single-nucleotide variants (SNVs) and insertions-deletions (INDELs) were identified based on the filtered variants with a mapping quality score ≥ 20 using the Genome Analysis Toolkit (GATK, version 3.6) software from the Broad Institute. The ANNOVAR software was used to functionally annotate variants.

Each FLC was picked out from the plate and lysed in 1 ml TRIzol reagent (Invitrogen, 15596026). 100 μl BCP (MRC, BP151) was added into the lysate, vortexed for 15 s, waited for 3 min at RT, then centrifuged for 15 min at 16,000 × g at 4 °C. The supernatant was transferred into a new tube followed by adding one volume of isopropanol and 20 μg glycogen, then precipitated at −20 °C overnight. The total RNA was centrifuged at 16,000 × g for 15 min at 4 °C, the precipitate was washed twice by 75% ethanol, then dissolved in RNase-free water. A total amount of 125 ng RNA of each sample was used for RNA-Seq. DNA library was prepared according to the protocol of NEBNext Ultra DNA Library Prep Kit for Illumina (NEB, E7370). The final quality-ensured libraries were pooled and sequenced on the Illumina HiSeq2000/2500 sequencer for 100 or 150 bp paired-end sequencing44 (link).