An. gambiae small RNA 24h post P. berghei infection

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 24h after P. berghei infected or control feeding were homogenized in TRIzol (Ambion). Total RNA was extracted by following standard manufacturer instructions. Small-RNA librairies were prepared from the small RNA fraction using TruSeq Small RNA Library reagents (Illumina). Libraries were sequenced using the Illumina Hiseq 2500 in a multiplexed 51 +7 bases single read using a TruSeq SR cluster kit v3 cBot HS, and a TruSeq SBS kit v3 HS 50 cycles (Illumina). Primary analysis of the sequences was performed with Casava software (v1.8 Illumina). Library preparation and sequencing was performed by ARK Genomics at the Roslin Institute (University of Edinburgh).