title: Comparative gene expression analysis of chloroquine sensitive and resistant strains of Plasmodium falciparum using RNA-Seq: Pfal3D7
design: P. falciparum chloroquine sensitive strain 3D7 were maintained in continuous culture by in vitro method, in ‘O’ positive red blood cells at 1-5 % parasitemia and 5 % haematocrit in RPMI 1640 (Gibco) supplemented with 25 mM HEPES buffer, 2.1 g/L sodium bicarbonate, 200 µg/ml gentamycin, 5 % albumax (Gibco) and 0.05 % hypoxanthine (Sigma). The parasite cells were harvested at 48 hr having ~ 5 % parasitemia with 90 % trophozoites. Total RNA was extracted from the infected red blood cells using Trizol method and mRNA purification was done by magnetic bead method.   cDNA synthesis was performed using reverse transcriptase and random primer and double strand cDNA was purified by AMPure XP beads. End repair was carried out to convert the overhangs into blunt ends and 3' adenylation prevent ligation to one another during adapter ligation reaction. Adaptor ligation was performed to ligate multiple indexing adapters to the ends of ds cDNA and helps in the hybridization onto flow cell. cDNA fragments were enriched using PCR which selectively enrich fragments having adapter on both ends and amplify amount of DNA in library. RNA-Seq was performed using Illumina HiSeq 2500 platform with paired-end reads to obtain 2 x 100 bp reads.