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Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg

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Namespace:Centre for Structural Systems Biology (CSSB), DESY and European Molecular Biology Laboratory Hamburg

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Protocols: The Plasmodium falciparum clone 3D7 was cultivated at a hematocrit of 5% in human O+ erythrocytes according to standard procedures (Trager, W. & Jensen, J. 1976. Human malaria parasites in continuous culture. Science 193:673-675. doi: https://doi.org/10.1126/science.781840). To obtain tightly synchronized parasites, cell cultures were synchronized twice with a delay of 6 hours and the resulting schizonts were separated using a magnetic MACS column system. Uninfected O+ erythrocytes were added to allow reinvasion during subsequent cultivation. After 8 hours, the culture was synchronized with 5% sorbitol (Lambros, C. & Vanderberg, J. P. 1979. Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65:418-420. doi: https://doi.org/10.2307/3280287), which allows only ring stage parasites to survive (4 hpi ± 4). Samples for RNA-seq were taken by centrifugation (800 x g) of 10 - 40 mL of P. falciparum cell culture after the next reinvasion at 8, 16, 24, 32, 40, 44 and 48 hpi to avoid influence of sorbitol treatment on gene expression. Additionally, merozoites were purified mechanically through filtration after 52 hours (Boyle, M. J. et al. 2010. Proc Natl Acad Sci USA 107:14378-14383. doi: https://doi.org/10.1073/pnas.1009198107). Red blood cell pellets were rapidly lysed in 10 - 20x volume pre-warmed guanidinium thiocyanate (TRIzol, Life Technologies) and stored at -70°C until RNA purification. The time course experiment was repeated thrice at an interval of 4 weeks to obtain three independently cultivated biological samples for RNA-seq. RNA was extracted using a silica-based purification protocol using the PureLink RNA Mini Kit (Life Technologies) in conjunction with TRIzol Reagent according to the manufacturer’s instructions and including DNase treatment on the column. Human globin mRNA was depleted from the samples using the GLOBINclear kit (Life Technologies) according to the manufacturer’s instructions. RNA quantity and quality were assessed using the Qubit RNA HA Assay kit with a Qubit 3.0 Fluorometer (ThermoFischer Scientific) and the Agilent RNA 6000 Pico Kit with an Agilent Bioanalyzer 2100 device. RNA-seq libraries were constructed using magnetic beads with oligo(dT) to enrich mRNA that was fragmented into short fragments using chemical fragmentation (high temperature and high salt) with PCR amplifier according to TruSeq RNA Sample Preparation v2 Guide (Part # 15026495 Rev. A). Then cDNA was synthesized using the mRNA fragments as templates. Short fragments were purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition for ligation of sequencing adapters. Fragments between 100-500bp (main peak at 130-160bp) were selected and cDNA libraries were amplified according an optimized protocol using KAPA HiFi polymerase in presence of the PCR additive TMAC to avoid sequencing bias caused by the high AT base composition content of P. falciparum (Oyola, S. O. et al. 2012. Optimizing illumina next-generation sequencing library preparation for extremely at-biased genomes. BMC Genomics 13:1. doi: https://doi.org/10.1186/1471-2164-13-1). During quality-control steps, Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR System were used for quantification and qualification of the sample library. The final library was quantitated in two ways: the average molecule length was determined using the Agilent 2100 bioanalyzer instrument (Agilent DNA 1000 Reagents), and the library was quantified by real-time quantitative PCR (QPCR) (TaqMan Probe).