Single-cell RNAseq library preparation from malaria parasite-infected RBCs

To prepare the single cell RNAseq libraries, we needed to enrich the parasite infected RBC’s thus increasing chances of loading and capturing mRNA transcripts from the parasites. Our unsynchronized parasite cultures were enriched using SLOPE as previously described^{34 (link),35 (link)} with some modifications. Briefly, erythrocyte density was measured using a Cellometer Auto T4 (Nexcelom Biosciences, Lawrence, MA) and cell density adjusted to 2×10⁹ erythrocytes/mL. The 22.5Units of the lytic agent streptolysin-O (SLO) per 50μl RBCs was added in a ratio of 2 parts SLO solution to 5 parts erythrocytes. Samples were mixed well by pipetting and incubated at room temperature for 6 min. 10 volumes of 1X PBS or media (RPMI 1640 HEPES) were added and cells were centrifuged at 2,500xg for 3 min. After removal of the supernatant, cells were washed twice more with 1X PBS. Following SLO lysis, cells suspended in 1X PBS were layered onto a 60% Percoll gradient (Sigma Aldrich, St Louis, MO) and centrifuged at 1,500xg for 10 min. The top layer of Percoll was discarded while the lower cell pellet, rich in infected erythrocytes, was collected and washed twice with 1xPBS and later suspended in 200μl RPMI to be loaded into the seqwell arrays.