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 determination of polyadenylation addition sites, polyadenylated RNA purified from total RNA was converted to cDNA using a 5’-T15VN-3’ oligonucleotide (V=A, G or C; N=T, A, G or C) primer to enrich for 3’ ends of transcripts. The cDNA was 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