mRNA stability study of Toxoplasma gondii in standard and low iron conditions. Transcriptome analysis of Toxoplasma gondii in after treatment with actinomycinD in standard and low iron conditions

Human foreskin fibroblasts cells were pre-treated for 24 hours with 100uM deferroxamine (an iron chelator)before being infected with RH parasites. At 18 h post infection, 10 mg/ml Actinomycin D was added to the culture media at in the treatment dishes. Parasites were collected at 0, 1, 3 and 5 hours post-Actinomycin D treatment. At each collection, host cells were mechanically lysed and filtered through a 3mm membrane to remove debris. Parasites were pelleted by centrifugation and immediately frozen on dry ice for storage at -80°C. RNA was extracted using a Qiagen RNAeasy kit according to manufacturer’s instructions. RNA libraries were then prepared using Illumina Stranded mRNA library preparation method and sequenced at 2 x 100 bp to an average of more than 10 million reads per sample.