title: PARA_3
organism: Anopheles coluzzii
description: Protocols: Three-day old female females Anopheles coluzzii mosquitoes were injected with dsPara or dsGFP, and after 4 days, total RNAs from three biological replicates were extracted and sequenced. For each biological replicate, pools of ~18 mosquitoes were extracted per treatment group. The para gene (AGAP004707), located on chromosome arm 2L, is composed of 39 exons, which transcribe 13 different messenger RNAs (mRNA) through alternate splicing. To maximize the efficacy of the gene silencing, two double stranded (ds) RNA for para were designed and synthesized for targeting each two distinct regions along the para gene with the aim to recognize all 13 transcripts. Each of the dsPara and the dsGFP used as control were prepared using the MEGAscriptTM RNAi Kit (Invitrogen by Thermo Fisher Scientific). Primers bearing the T7 RNA polymerase recognition sequence were designed to amplify ~604 (Para1) and ~730 (Para2) base pairs regions. For gene silencing procedure, one to three day old cold-anaesthetized mosquitoes were injected using the NanoJect II (Drummond) either with the equimolar mix of the two dsPara or with dsGFP for the control group. The mosquitoes were all injected into the thorax with approximately 1ug of dsRNA and maintained on sugar water until required for downstream experiments. Using Illumina's Truseq RNA Sample Preparation Kit 1 microgram of total RNA was oligo-dT purified using oligo-dT coated magnetic beads, fragmented and then reverse transcribed into cDNA. The cDNA was fragmented, blunt-ended, and ligated to indexed (barcoded) adaptors and amplified using 15 cycles of PCR. Final library size distribution was validated using capillary electrophoresis and quantified using fluorimetry (PicoGreen) and via q-PCR. Indexed libraries were then normalized, pooled and then size selected to 320bp +/- 5% using Caliper's XT instrument. Truseq libraries were hybridized to a paired end flow cell and individual fragments were clonally amplified by bridge amplification on the Illumina cBot.