title: Illumina HiSeq 2000 paired end sequencing; RNA-seq of Leishmania mexicana promastigotes, axenic amastigotes and intracellular amastigotes
design: RNA-seq of Leishmania mexicana promastigotes, axenic amastigotes and intracellular amastigotes
library_construction_protocol: Promastigote-form L. mexicana (WHO strain MNYC/BZ/62/M379) were grown at 28°C in M199 medium (Life Technologies) supplemented with 2.2 g/L NaHCO3, 0.005% haemin, 40 mM 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) pH 7.4 and 10% FCS (Life Technologies 10500-064). Cells were maintained in exponential growth phase 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.
attributes.tag: Experimental Factor: organism
attributes.value: Leishmania mexicana
attributes.tag: Experimental Factor: developmental stage
attributes.value: promastigote
attributes.tag: Experimental Factor: host
attributes.value: not applicable (outside host)