Table 1. Reagents permuted during optimisation of the single cell RNAseq protocol and stats of each treatment condition after sequencing.
Different combinations of the protocol were tested by sequencing. Initial trials were performed with 2 µl of lysis buffer, this was increased to 4 µl to augment capture efficiency. Permutations of the protocol that were tested were a terminal anchoring base (A,G,C; V) or not (T), two reverse transcriptase enzymes (Smartscribe (SmSc); Superscript II (SII)) and 25 or 30 cycles of preamplification. Both sexual and asexual cells of P. berghei and P. falciparum were tested. For each sequenced dataset, we calculated the mean percentages of rRNA, mRNA and other reads across the cells. For some samples we also downsampled the data to 50,000 reads per cell to allow comparison of the number of genes detected. This was done to determine differences in the complexity of each library. For the three larger datasets produced (P. falciparum gametocytes, P. berghei mixed blood stages, and P. falciparum asexual stages), we provide the numbers of pre- and post-filtered cells and median number of genes in those filtered cells.
| Conditions tested | Protocol | SSII, V30, 30 cycles | SSII, T30, 30 cycles | SmSc, T30, 30 cycles | SSII, T30, 25 cycles | SmSc, T30, 25 cycles | SmSc, T30, 25 cycles | SmSc, T30, 25 cycles | SmSc, T30, 25 cycles |
|---|---|---|---|---|---|---|---|---|---|
| Cells | Sexual | Asexual | Asexual | Asexual | Asexual | Sexual | Mixed blood | Asexual | |
| Species | Pf | Pf | Pf | Pf | Pf | Pf | Pb | Pf | |
| Lysis buffer volume | 2 µl | ✓ | |||||||
| 4 µl | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ||
| Oligo Dt (IDT) | Anchored 30 bp | ✓ | |||||||
| Non-Anchored 30 bp | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ✓ | ||
| Reverse transcriptase | Superscript II (Life Technologies) 10U | ✓ | ✓ | ✓ | |||||
| Smartscribe (Clontech) 5U | ✓ | ✓ | ✓ | ✓ | ✓ | ||||
| Cycle number | 25 | ✓ | ✓ | ✓ | ✓ | ✓ | |||
| 30 | ✓ | ✓ | ✓ | ||||||
| Sequencing machine | HiSeq | ✓ | ✓ | ✓ | |||||
| MiSeq | ✓ | ✓ | ✓ | ✓ | ✓ | ||||
| Sequencing results summary | % rRNA | 5.7 | 33.5 | 36.2 | 6.4 | 18.4 | 17.8 | 16.7 | 34.8 |
| % coding genes | 4.4 | 11.3 | 39.3 | 10.5 | 33 | 51.7 | 49 | 40.5 | |
| % other | 90 | 55.2 | 24.4 | 83.1 | 48.6 | 30.5 | 34.2 | 24.6 | |
| Median genes detected for 50k reads | 25 | 84 | 145 | 174 | 181 | 502.5 | NA | NA | |
| Total cells | 5 | 6 | 6 | 6 | 6 | 237 | 182 | 174 | |
| Cells passing filters | NA | NA | NA | NA | NA | 191 | 144 | 161 | |
| Median gene count | NA | NA | NA | NA | NA | 2011 | 1922.5 | 1793 |