Trypanosoma vivax Y486 bloodstream stage RNAseq
Trypanosoma vivax Y486 bloodstream stage RNAseq
Trypanosoma vivax Y486 was grown from stabilate in BALB/c mice immunosuppressed with cyclophosphamide (250 mg.kg-1) and was amplified at patent parasitaemia in three immunosuppressed mice, from which whole blood was collected. The blood was treated with the erythrocyte lysis buffer (EL; QIAGEN), following the manufacturer's instructions, and RNA was isolated from the pellet using the RNeasy mini kit protocol (QIAGEN). Poly A+ mRNA was purified from total RNA using oligo dT dyna bead selection followed by metal ion hydrolysis fragmentation with the Ambion RNA fragmentation kit. 1st strand cDNA was synthesized using randomly primed oligos followed by 2nd strand synthesis to produce dscDNA. This was followed by end repair with T4 and Klenow DNA polymerases and T4 polynucleotide kinase to blunt-end the DNA fragments. A single 3' A nucleotide was added to the repaired ends using Klenow exo- and dATP to deter concatemerization of templates, limit adapter dimers and increase the efficiency of adapter ligation. Adapters were then ligated (containing primer sites for sequencing). Ligated fragments were run on an agarose gel, size selected for 275-325bp inserts and DNA extracted as per Qiagen gel extraction kit protocol but with dissolution of gel slices at room temperature (rather than 50oC) to avoid heat induced bias. Libraries were then amplified by PCR to enrich for properly ligated template strands, to generate enough DNA and to add primers for flowcell surface annealing followed by a clean up with AMPure SPRI beads. These libraries were quantified using Agilent Bioanalyser chip and Kapa Illumina SYBR Fast qPCR kit.
Experimental Factor: StrainOrLine
Y486