| description |
The A. gambiae s.s. Ngousso colony was originally initiated with mosquitoes collected in Yaoundé, Cameroon in January 2006 (Harris et al., 2010), and belongs to the M molecular and Forest chromosomal forms. The strain was reared in the insectary of the Pasteur Institute CEnter for Production and Infections of Anopheles (Platform CEPIA, Institut Pasteur, France) under standard rearing conditions at 26°C and 80% relative humidity, under a 12 h light/dark cycle as previously described (Harris et al., 2010). Mosquitoes were fed on mice infected with a clone of P. berghei strain pbANKA, expressing GFP under the hsp70 promoter at 5-10% parasitemia with mature gametocytes, gametocyte maturity was tested by exflagellation of microgametes. Mosquitoes were maintained at 21°C and 80% relative humidity on 10% sucrose. Pools of 30 An. gambiae Ngousso mosquito midguts 48h after P. berghei infected or control feeding were homogenized in TRIzol (Ambion). Total RNA was extracted by following standard manufacturer instructions. Immunoprecipitation was performed on the extracted RNA using protein G agarose (Invitrogen) and anti-dsRNA antibody J2 (scicons). 3 conditions were performed: RNA from infected midguts and the J2 antibody; RNA from infected midguts and a mouse IgG2a isotype control; RNA from uninfected midguts and the J2 antibody. Biological duplicates were performed. Bound RNA on the beads was extracted using Trizol (Ambion).Paired end librairies then were prepared using Paired end RNA Library reagents (Illumina). Libraries were sequenced using the Illumina Hiseq 2500 in a HiSeq Rapid Run worflow (Illumina). Primary analysis of the sequences was performed with Casava software (v1.8 Illumina). Library preparation and sequencing was performed by the Institut Pasteur Transcriptomics and Epigenomics core facility. |