GSM5456749: TOP03-PcyM-D2a; Plasmodium cynomolgi; RNA-Seq
Sorting was performed using a 100 µm nozzle at 20 PSI or with a 130 µm nozzle at 11.5 PSI. GFP positive parasites (see gating strategy in article) were collected in 384 wells plates containing 100 nl primer and 5ul mineral oil with the sorting precision at “single cell” (yield mask: 0; purity mask: 32; phase mask: 16) to maximize the purity and counting accuracy. By including the index sorting function, information on parameters such as forward scatter (FSC), side scatter (SSC), GFP and mCherry signals was recorded for each well containing a sorted event. In this way, information could be traced back to specific wells of interest. As controls, single cell sorts were performed through gating on uninfected hepatocytes (performed for every plate), and by gating on GFPpositive/mCherry positive parasites  and GFP positive/mCherry negative parasites (only in batch 1, day 6). Immediately after sorting, plates were centrifuged (2 min., 350 g, 4 C) and stored at -80 C until further processing. Cel-seq2 protocol (Tamar Hashimshony et al, 2016). In short, single cells were sorted based on their GFP signal into 384-well plates containing 100 nl of unique CEL-seq2 compatible primer (7 ng/µl) and 5 µl mineral oil (Sigma-Aldrich). Following cell lysis and denaturation, reverse transcription was performed using 3.5 units SuperScript II reverse transcriptase per well (Invitrogen, #18064014) in the presence of 20 nl ERCC spike-in per well (Ambion™ 4456740, 1:5000 diluted in H2O). Single-cell RNA-seq libraries were sequenced for 2x 42 bp on the Illumina NextSeq 500 system (read 1 is part of the 3' end mRNA, read 2 contains the UMI and cell barcode).
GEO
GSM5456749