| description |
Recent advances in high throughput sequencing methodologies allow the opportunity to probe in depth the transcriptomes of organisms including important human pathogens. In this project, we are using Illumina sequencing technology to analyze the transcriptome (RNA-Seq) of experimentally accessible stages of the mouse malaria parasite, P. chabaudi AS. The aim is to analyse cir gene expression during Plasmodium chabaudi infection and determine whether host genetic background can influence cir expression.This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/.Protocols - Female BALB/c and C57BL/6 mice aged 6–8 weeks were obtained from the specific pathogen-free unit at the MRC National Institute for Medical Research (NIMR), London. For experimental purposes, mice were housed conventionally with sterile bedding, food and irradiated water on a 12 hour light-dark cycle. A cloned line of Plasmodium chabaudi chabaudi (AS) was used in this study. Stabilates were cryo-preserved in blood from BALB/c mice. To obtain parasites for experimental infection, an aliquot of the stabilate was injected intraperitoneally (i.p.) into an immunodeficient BALB/c RAG2-/- mouse. Blood was taken from the donor mice 7 days after infection and experimental BALB/c or C57BL/6 mice were infected by injecting 105 infected erythrocytes i.p. Parasitaemia was monitored by examination of Giemsa-stained blood films as previously described. Blood was collected from each mouse by cardiac puncture under terminal anaesthesia into Krebs saline (114 mM NaCl, 4.57 mM KCl, 1.15 mM MgSO4) containing 0.2% glucose and 25 U/ml heparin (Leo Pharmaceuticals) 7 days after infection. Leukocytes were removed via Plasmodipur filtration (Euro-Diagnostica) according to manufacturer’s instructions. Blood was then stored at -80 °C in TRIZOL reagent (Invitrogen) for subsequent RNA extraction.RNA was extracted from P. chabaudi infected blood samples by guanidinium thiocyanate-phenol-chloroform extraction according to standard methods and DNase digested using Turbo DNAse (Ambion) according to the manufacturer’s instructions. RNA was depleted of ribosomal RNA with exonuclease followed by purification with oligo dT and libraries were created using the Illumina RNA-seq protocol.The samples were sequenced on either the Illumina Genome Analyser II or Illumina HiSeq 2000 following standard manufacturers protocols. |