Plasmodium yoelii RNA-Seq
Plasmodium yoelii RNA-Seq
Groups of 5 Balb/c mice were infected i.v. on day 0 with either 200, 1000, or 5000 WT or PY01365-KO parasitized erythrocytes. Blood smears from each mouse were made daily from D3, stained with Giemsa's reagent and infected cells counted to monitor the course of infection. Erythrocytes containing late stage parasites, were harvested at 20 to 25(nil)arasitaemia and schizonts were purified by centrifugation for 25 min at 600 g at room temperature (RT) on a 55% Nycodenz (Nycoprep) cushion (NYCOMED Pharma AS). 5_107 schizonts were mixed with 90 ul AMAXA nucleofactor T-cell solution (plus supplements) and 5 ug of targeting construct DNA was added. These parasites were transfected using AMAXA Nucleofector programme U33. Immediately, 100 ul of RPMI 1640 medium containing 20 0.000000oetal calf serum (FCS) was added to the transfected parasites and the suspension injected intravenously (i.v.) into the tail vein of a single mouse. Electroporation of parasites with targeting construct and injection into individual mice was carried out twice independently using the above conditions. Pyrimethamine-resistant parasites were verified by PCR and Southern blot analysis and the transgenic parasite line was cloned by limiting dilution. Parasites were purified using a MACS type-D depletion column with a SuperMACS II magnetic separator (Miltenyi Biotec GmbH). Total RNA was prepared from leukocyte-depleted, Percoll-purified late stage WT and PY01365-KO parasitized erythrocytes using Trizol (Invitrogen Life Technologies). RNA samples were first treated with RNase-Free DNase I (Quiagen) and cleaned up using RNeasy MiniElute to remove contaminating gDNA. RNA of both samples was depleted of ribosomal RNAs with exonuclease and  and libraries were created using the Illumina RNA-seq protocol.
Experimental Factor: GENOTYPE
wild_type