GSM9110396: Mini-bulk samples indexed with RPI-48; Plasmodium falciparum; RNA-Seq
At 30 hours, cultures with and without DFO were treated with saponin to obtain parasite pellets. Pellets from each treatment condition were resuspended in 50uL of RNAlater and stored at -20°C for RNA extraction and sequencing. Four biological replicates were performed at least one week apart, and all samples from the replicates were subsequently processed together. RNA was extracted using the Isolate II RNA mini kit (Bioline) and performed as per the protocol and eluted in 35uL of water. RNA was quantified using the Qubit 2.0 HS RNA assay (Thermo Fisher) and the quality of a subset assessed using the 2200 TapeStation System High Sensitivity RNA Screen Tape (Agilent). Samples were normalised to RNA concentrations of 10ng/mL. Sequencing was performed using WEHI's Cellular Genomics Projects Team's mini bulk sequencing protocol. In short, single cell transcriptome libraries were generated by adapting the CelsSeq2 protocol as follows: samples were pooled after first strand cDNA synthesis, treated with Exonuclease 1 for 30 minutes, followed by a 1.2X bead clean-up. Second strand synthesis was performed using NEBNext Second Strand Synthesis module (NEB) in a final reaction volume of 20 µL and NucleoMag NGS Clean-up and Size select magnetic beads (Macherey-Nagel) were used for all DNA purification and size selection steps. Sequencing was performed on the Illumina NextSeq2000 instrument (San Diego, USA), using a 100 cycle kit.