title: Bulk RNA-seq of the human malaria parasite Plasmodium falciparum 3D7 cultured under different iron conditions
design: Bulk RNA-seq of the human malaria parasite Plasmodium falciparum 3D7 cultured under different iron conditions
library_construction_protocol: Samples for RNA-sequencing were prepared in triplicate for each condition and time point, i.e. three separate parasite cultures each were grown in parallel for over two weeks. During the second intra-erythrocytic developmental cycle after reinvasion into erythrocytes from the respective donor, two 10-mL dishes each were harvested for parasites at the ring stage (6 – 9 hpi) and one 10-mL dish each for trophozoites (26 – 29 hpi). Samples were collected by centrifuging the culture for 5 min at 800 g and 37°C and dissolving the erythrocyte pellet in 5 mL TRIzol prewarmed to 37°C, followed by immediate transfer to -80°C for storage. The parasitemia was 0.3% at the start of the experiments with high, medium and low-iron donor blood and 2 – 3% at the time of harvest. Cultures treated with hepcidin had a starting parasitemia of 0.6% and those without hepcidin 1% to reach a parasitemia of 4 – 5% during the second cycle. For each experiment, the parasitemia was kept consistent at the point of harvest as high parasite densities can affect transcription. The P. falciparum strain 3D7 was cultured according to modified standard procedures at 5% hematocrit using human 0+ erythrocytes from University Medical Center Hamburg-Eppendorf (UKE) at 1% O2, 5% CO2 and 94% N2. RPMI 1640 medium was supplemented with 0.5% (w/v) AlbuMAX II, 20 µg/mL gentamicin and 100 µM hypoxanthine (Thermo Fisher Scientific).  For experiment 1, erythrocytes from an individual with high iron status (231 µg/L ferritin, 18.2 g/dL Hb, 51.5% hematocrit), another with iron deficiency (3 µg/L ferritin, 11.4 g/dL Hb, 36.3% hematocrit) and a healthy individual (21 µg/L ferritin, 15.0 g/dL Hb, 42.3% hematocrit) were used. For experiment 2, red blood cells from another healthy individual with or without the addition of 0.7 µM hepcidin (Bachem) to the culture medium was used for comparison. Parasites were synchronized to a 3-h age window by isolating late schizonts from a 60% Percoll (GE Healthcare, Life Sciences) gradient and culturing these for 3 h with erythrocytes from the respective donor, followed by controlled elimination of advanced parasite stages using 5% (w/v) D-sorbitol (Carl Roth) for 10 min at 37°C. For RNA extraction, the samples frozen in TRIzol were thawed, mixed thoroughly with 0.1 volume cold chloroform, and incubated at room temperature for 3 min. Following centrifugation at 20,000 g and 4°C for 30 min, the supernatants were transferred to fresh vials and combined with an equal volume of 70% ethanol. RNA was purified using the RNeasy MinElute Kit (Qiagen) by on-column DNase I digest for 30 min and elution with 14 µL water. The GLOBINclear Human Kit (ThermoFisher Scientific) was then employed to deplete human globin mRNA in all samples. The Qubit RNA HS Assay Kit and Qubit 3.0 fluorometer (ThermoFisher Scientific) were used for RNA quantification. Upon arrival at the EMBL Genomics Core Facility (GeneCore Heidelberg, Germany), the RNA quality of each sample was evaluated using the RNA 6000 Nano kit and Bioanalyzer 2100 (Agilent). The median RNA integrity number (RIN) of all samples was 7.30 (IQR: 6.85 – 8.15). Individually barcoded strand-specific libraries for mRNA sequencing were prepared from total RNA samples of high quality (approximately 150 ng per sample) using the NEBNext® RNA Ultra II Directional RNA Library Prep Kit (New England Biolabs) for 12 PCR cycles on the liquid handler Biomek i7 (Beckman Coulter GmbH) at GeneCore.