Name: Sample 11_s
Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: size fractionation
Layout: SINGLE
Construction protocol: 12 mosquitoes per pool, in each of two biological replicate pools, were collected 3 d after infection by oral feeding with ONNV and homogenized in TriReagent (Sigma). The A. coluzzii Ngousso strain was originally initiated with mosquitoes collected in Cameroon in January 2006, and belongs to the M molecular and Forest chromosomal forms (Harris et al., 2010). Mosquitoes were reared under standard conditions at 26°C and 80% relative humidity, with a 12 h light/dark cycle as previously described (Mitri et al., 2015). Double-stranded RNAs were synthesized from PCR amplicons using the T7 Megascript Kit (Ambion) as described (Carissimo et al., 2015). For each targeted gene, 500 ng of dsRNA was injected using glass capillary needles into the thorax of cold-anesthetized 1–2 d-old A. coluzzii females using a nano-injector (Nanoject II, Drummond Scientific). Mosquitoes were infected on an infectious bloodmeal containing physiologically natural viral titers O'Nyong-Nyong strain SG650. 10ug of RNA (>200nt fraction) was used to prepare directional libraries using the TruSeq SmallRNA sample prep kit, set A and B (Illumina). Chemical fragmentation of polyA RNA was made with Ambion reagent (AM8740), followed by purification on RNeasy columns (Qiagen, #74204). Phosphatase and PNK treatments were performed and followed by purification on RNeasy columns (Qiagen, #74204). The fragmented RNA was ligated with 3'- and 5'- TruSeq adapters, as described in the original protocol. Synthesis of cDNA was performed by reverse transcription. The cDNA product was then specifically amplified by 11 cycles of PCR and products purified on Agencourt AMPure XP beads (Beckman Coulter Genomics, # A63881).