Illumina Genome Analyzer II paired end sequencing; Schistosoma mansoni transcriptomics at different life stages
Schistosoma mansoni transcriptomics at different life stages
A  Peurto Rican strain of S. mansoni was used in this study.  Mixed-sex worms (including female worms) were perfused from percutaneously infected Golden Syrian hamsters challenged 7-weeks earlier with cercariae and provided by Dr. Phil LoVerde (University of Texas, San Antonio, Texas, U.S.). Cercariae were shed from Biomphalaria glabrata intermediate snail hosts. Mechanically transformed schistosomula were prepared by mechanical transformation of cercariae and cultured at 37C in DMEM (Sigma, UK) supplemented with 10 0.000000oetal calf serum, 2 mM L-glutamine, and 100 ug/ml penicillin/streptomycin in an atmosphere of 5CO2 for 3hr or 24hr. Skin transformed schistosomula were prepared by allowing cercariae to penetrate a layer of freshly excised mouse skin. After penetration, schistosomula were cultured at 37C in DMEM (Sigma, UK) supplemented with 10 0.000000oetal calf serum, 2 mM L-glutamine, and 100 ug/ml penicillin/streptomycin in an atmosphere of 5CO2 for 24hr. Cercarial tails were obtained using the same procedure as for mechanically transformed schistosomula. (Parameters: time unit = seconds, temperature unit = C) S. mansoni total RNA was extracted from parasites homogenised in TRIzol reagent (Invitrogen UK) using an Ultra-Turrax T10 dispersing tool (IKA-Labortechnik, Janke & Kunkel, GmbH & Co). Cercariae total RNA was isolated using a modified TRIzol/RNeasy (Invitrogen UK; Qiagen UK) procedure as previously described, RNA from the rest of the samples was extracted using  TRIzol(Invitrogen, UK)according to manufacturers instructions. All RNA was subsequently quantified with a NanoDrop ND-1000 UV-Vis spectrophotometer and visualized for quality using an Agilent 2100 bioanalyzer. (Parameters: Extracted product = total_RNA, Amplification = none)
Experimental Factor: DEVELOPMENTALSTAGE
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