title: Illumina HiSeq 2500 sequencing; RNA-seq of Spodoptera frugiperda Sf9 cell line
design: RNA-seq of Spodoptera frugiperda Sf9 cell line
library_construction_protocol: Sf9 cells are grown in F75 plates. They were seeded with 3E5 cells/mL/well. When confluence is reached, the medium was removed from the plates and cells were rinsed with cold 1X PBS. Cells were then scraped from the plate in 1mL 1X cold PBS. Cells were centrifuged for 2 minutes at 1500 rpm at 4°C. The supernatant was removed and the pellet resuspended in 300µl of TE buffer supplemented with Complete Mini Protease inhibitor cocktail. Cells were lyzed by sonication with a Diagenode Bioruptor (3 cycles, 30\" ON; 30\" OFF). Sf9 cells from ATCC are cultured at 28°C according to manufacturer recommandations. \"The base medium for this cell line is Grace's Insect Medium Supplemented (GIBCO/Invitrogen Cat. No. 11605-094 or equivalent). To make the complete growth medium, add the following components to the base medium: heat-inactivated insect cell culture tested fetal bovine serum (Sigma cat# F4135) to a final concentration of 10%.\" RNA was extracted with the RNEasy kit from Qiagen and treated with turboDNAse (Ambion). Total RNA was used for RNA-seq libraries. Library preparation and RNA sequencing were conducted by MGX GenomiX (IGF, Montpellier, France). Libraries were prepared with the TruSeq Stranded mRNA Sample preparation kit (Illumina). In brief, after a purification step with oligo(dT) magnetic beads, polyadenylated RNAs were chemically fragmented. A first cDNA strand was synthesized with random primers and SuperScript IV Reverse Transcriptase (Life Technologies) and the second strand was then synthesized. After the addition of single adenine nucleotides, indexed adapters were ligated to the cDNA ends. Adapters-ligated cDNAs were then amplified by PCR and libraries were validated on Fragment Analyzer with a Standard Sensitivity NGS kit (Advanced Analytical Technologies, Inc) and quantified by qPCR with a Light Cycler 480 thermal cycler (Roche Molecular diagnostics).