GSM5737643: 16 D; Plasmodium falciparum; RNA-Seq
For 40 h.p.i. time point tight synchronous parasites were enriched using 63% Percol, washed twice with incomplete RPMI media and processed immediately on the 10X Chromium controller (10X Genomics, Pleasanton, CA). For 16 h.p.i. time point parasites were stained with Mitotracker Deep Red FM (Life Technologies, #M22426) for FACS analysis and flow sorting respectively. Briefly, 50 µl of SYBR Green I stained RBC's were analyzed on BD LSR Fortessa Flow Cytometer with High Throughput sampler (BD Biosciences, San Jose, CA, USA) using BD FACS Diva Software v6.2 and 488 laser excitation / 530 emission filter to determine concentration of SYBR Green I positive cells per microliter. A BD Influx Cell Sorter (BD Biosciences, San Jose, CA, USA) with BD FACS Software v1.0.01 software was used to sort ~40,000 MitoTracker Deep Red FM - positive RBC's using a 70 µm nozzle, a 640 nm laser excitation / 670 nm emission filter and a pressure setting of 30 psi. Post-sorted cell concentration and quality were checked using a Countess® II Automated Cell Counter (Invitrogen) and FLoid™ Cell Imaging Station (ThermoFisher). Finally, labelled cells (i.e. SYBR Green I or MitoTracker Deep Red FM positive cells) were then loaded onto a 10X chip (Chip G) and processed immediately on the 10X Chromium controller (10X Genomics, Pleasanton, CA). Single cell libraries were constructed using the 10X Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 with Single Index Kit T Set A. Due to the extremely low RNA content of single cell malaria parasite's and the AT rich genome (~70% AT), modifications to the cDNA amplification and library preparation workflow were made accordingly. These modifications included - 30x cDNA amplification cycles; taking 50% cDNA as input into library generation; reducing fragmentation time to 2 minutes; and changing the extension time to 65 oC during index PCR. Individual library Qc was performed using the BioAnalyzer HS DNA Assay kit (Agilent).