Illumina HiSeq 4000 paired end sequencing; Different PfEMP1-expressing Plasmodium falciparum variants induce divergent endothelial transcriptional responses during cytoadherence
Different PfEMP1-expressing Plasmodium falciparum variants induce divergent endothelial transcriptional responses during cytoadherence
At each time point, the co-culture medium was removed, and the cells were washed once with EC medium to remove unbound IE or uninfected RBC. 1.5 ml of EC medium was added to each flask prior to harvesting the cells by cell-scraper. The cells were transferred to 1.5 ml sterile tubes and stored at -80°C. Malaria parasite culture. Laboratory-adapted IT4var14 (A4) [PMID: 1614515] and IT4var37 (4E12) [PMID: 21573138] P. falciparum isolates were cultured at 2% haematocrit in O+ human erythrocytes in complete RPMI 1640 medium (supplemented with 10% human serum, 37.5 mM HEPES, 11 mM D-glucose, 25 mg/ml gentamicin sulphate, 2 mM L-glutamine, pH 7.4) [PMID: 30103779]. IT4var14 binds to ICAM-1 and CD36, whereas IT4var37 is a strong CD36-binder but does not bind to ICAM-1 [PMID: 21573138]. IT4var14 binds to HUVEC, HBMEC and HDMEC. P. falciparum undergoes antigenic variation in culture, which changes the proportion of IE expressing specific PfEMP1s in the parasite populations.  To minimize var gene switching, IT4var14 was selected with specific BC6 antibody [PMID: 1614515] coated protein G Dynabeads using standard protocols [PMID: 25360558].  IT4var37 does not have a specific antibody available for selection, but this variant is common [PMID: 21573138] and the presence of a significant proportion of this PfEMP1 expression was monitored by qRT-PCR using specific primers (ABRA6 – [PMID: 17878945]).  The selected and screened parasite strains were used for experiments for up to 3 weeks. The parasite lines, EC cultures, washed blood cells and parasite culture media were regularly monitored for mycoplasma contamination using the Universal Mycoplasma Detection Kit (ATCC® 30-1012K™). Endothelial Cell culturing. Primary human brain microvascular endothelial cells (HBMEC) were obtained from Cell Systems, USA (ACBRI 376). HBMEC were cultured in 1% gelatin-coated flasks in Promocell's Endothelial Cell Growth Medium MV (C-22020) supplemented with Endothelial Cell Growth Medium MV Supplement Mix (C-39225) and passaged using the Detach Kit (C-41220), following manufacturer's instructions.  Co-culture of EC with malaria parasite. HBMEC at passage 4 to 8 were cultured till 80-90% confluency in 25 cm2 flasks and, for +TNF conditions, stimulated with 10 ng/ml TNF overnight. Pf-IE were synchronised with 5% sorbitol, cultured until they reached 5-8% parasitaemia (mature stages) and enriched by Plasmion to obtain 50-60% parasitaemia. The enriched Pf-IE were resuspended in EC medium to a standard parasitemia of 50% at 1% haematocrit and overlayed onto the HBMEC for 0 (on-off), 2, 6 and 20 hours, in triplicate, in a 5% CO2 incubator. Uninfected red blood cells were used as a control. At each time point, the co-culture medium was removed, and the cells were washed once with EC medium to remove unbound IE or uninfected RBC. 1.5 ml of EC medium was added to each flask prior to harvesting the cells by cell-scraper. The cells were transferred to 1.5 ml sterile tubes and stored at -80°C. RNA was extracted using the RNeasy Mini Kit (Qiagen, 74104) following manufacturer's instructions. The quality of the RNA samples was assessed using an Agilent 2100 Bioanalyzer (RNA 6000 Nano Kit, 5067-1511, Agilent Technologies) following manufacturer instructions.  All RNA samples had a RIN of 8.5 or higher, indicating high-quality RNA. The quality of the RNA samples was assessed using an Agilent 2100 Bioanalyzer (RNA 6000 Nano Kit, 5067-1511, Agilent Technologies) following manufacturer instructions.  All RNA samples had a RIN of 8.5 or higher, indicating high-quality RNA. Strand-specific RNA-sequencing (ssRNA-seq) libraries were prepared using the Illumina Truseq Stranded mRNA Sample Preparation Kit (Illumina, RS-122-2101) following the manufacturer's instructions.