Illumina HiSeq 2000 paired end sequencing; RNA-seq of Leishmania mexicana promastigotes, axenic amastigotes and intracellular amastigotes
RNA-seq of Leishmania mexicana promastigotes, axenic amastigotes and intracellular amastigotes
To generate intracellular amastigotes, Leishmania mexicana promastigotes (WHO strain MNYC/BZ/62/M379) were left to grow into stationary phase (from 1x10E5 to 2x10E7 cells/ml) and then incubated with BMDMs at a parasite to macrophage ratio of 20 for 2h before washing off remaining extracellular parasites. Infected macrophages were incubated 24h at 34°C prior to RNA extraction. For mapping of splice acceptor sites and determination of transcript abundances, polyadenylated RNA was purified from total RNA, converted to cDNA using random hexamer primers, sheared and size selected for fragments ~200bp in length using the Illumina TruSeq RNA Sample Preparation Kit v2.
Experimental Factor: organism
Leishmania mexicana and Mus musculus
Experimental Factor: developmental stage
amastigote
Experimental Factor: host
Mus musculus