GSM7066359: 796_L2; Drosophila melanogaster; RNA-Seq
For each sample, we extracted RNA using phenol chloroform phase separation. We first added 500 µl of TRIzol (Thermo Fisher Scientific) to the frozen specimens and crushed them with a pestle. We then added 100 µl of chloroform to the crushed specimens, agitated the samples for 15 seconds, and incubated them at room temperature for 3 minutes. The samples were centrifuged at 12,000 RCF for 15 minutes at 4°C. We transferred the aqueous phase into fresh tubes, added an additional 250 µl of isopropanol, and inverted the tube four times to ensure mixing. After allowing the tubes to incubate at room temperature for 10 minutes, the samples were centrifuged at 12,000 RCF for 10 minutes at 4°C. We removed the supernatant, washed the pellet with 500 µl of 75% ethanol, and again centrifuged the samples at 7,500 RCF for 5 minutes at 4°C. A second wash was then carried out using 500 µl of 75% ethanol. After a final centrifugation at 7,500 RCF for 5 minutes at 4°C, the supernatant was carefully removed. The pellet was left to dry at room temperature for 30 min. RNA was resuspended in 100 µl of nuclease-free water and then the samples were heated to 55°C in a drybath for 15 minutes. We verified RNA concentration and integrity on the Bioanalyzer (Agilent) with the RNA 6000 Pico Kit according the manufacturer instructions, as well as on the NanoDrop™ 8000 Spectrophotometer (Thermo Fisher Scientific). All RNA samples were stored at -80°C. We prepared the cDNA libraries using the Illumina® TruSeq® Stranded mRNA Sample Preparation Kit, following the manufacturer's protocol and using a Biomek NXᴾ Automated Workstation (Biomek NXP Span-8 by Beckman). In brief, poly-A-containing mRNA molecules were purified from 1 µg total RNA using poly-T oligo-attached magnetic beads. The purified mRNA was fragmented by adding the fragmentation buffer and then heated to 94°C in a thermocycler for 8 minutes. We primed the cleaved RNA fragments with random hexamers and used reverse transcriptase to synthesize first-strand cDNA. Second-strand cDNA synthesis, end repair, A-tailing, adapter ligation, and enrich DNA fragments were carried out in accordance with the manufacturer supplied protocols. Each indexed cDNA library was verified and quantified using a dsDNA 915 Reagent Kit on a Fragment Analyzer (Agilent). We equally mixed 12 to 13 indexed cDNA libraries in each final library. The final libraries were quantified by real-time PCR using the KAPA Library Quantification Kit for Illumina Sequencing Platforms (Kapa Biosystems Ltd, SA), adjusted to 10 nM in water, and provided to the Montpellier Genomix platform (http://www.mgx.cnrs.fr/) for sequencing. Each final mixed cDNA library was denatured with NaOH and diluted to a concentration of 18 pM using the standard library PhiX (1%) from Illumina. Flow cell was clustered using TruSeq SR Cluster Kit v3-cBot-HS, and then loaded into the Illumina HiSeq 2500 instrument following the manufacturer's instructions. The v3 sequencing chemistry (TruSeq SBS Kit v3-HS) was used, applying a 50 cycle, single-read, indexed protocol. Image analyses and basecalling were carried out using the Hiseq Control Software (Illumina HCS) and Real-Time Analysis Software (Illumina RTA).