title: GSM9382187: wild type IGP2 and ∆GID1/2/7/8/9 gametocytes at day 3,6,9 and 12 of development.; Plasmodium falciparum; RNA-Seq
library_construction_protocol: Parasite cultures were harvested at the desired lifecycle stage, centrifuged at 500 x g and the supernatant discarded. Infected RBC pellets were then lysed with 5 pellet volumes of 0.03% saponin and incubated on ice for 15 minutes. The samples were centrifuged at 16,000 x g, the supernatants discarded, and the pellets washed 3 time with 1xPBS. From these pellets RNA was isolated from the PfGID1, 2, 7, 8 and 9 knockouts and parental iGP2 parasites at day 3, 6, 9 and 12 of gametocyte (five biological replicates for each cell line and timepoint were performed) development using the Isolate II RNA mini kit (Bioline). The RNA concentration of each sample was calculated using the QuantiFluor RNA system (Promega), and RNA quality was determined using high-sensitivity RNA screen tape analysis (Agilent). The RNA concentrations were then normalized to 1 ng/µL by dilution with the FlexDrop automated dispenser (Revvity), and then re-quantified. 1.1 ng of total RNA was reverse transcribed using SuperScript II (Invitrogen) and processed using an adapted CelSeq2 protocol. Briefly, barcoded samples with unique molecular identifiers (UMIs) were pooled after first strand cDNA synthesis and second strand synthesis performed using the MEGAscript T7 transcription kit (ThermoFisher). Amplified RNA was reverse transcribed using a tagged random hexamer without fragmentation, followed by sequencing on a NextSeq 2000 P3 flow cell (Illumina). Technical replicates were performed for each RNA sample.