| Growth Protocol: |
Parasites were cultivated in type O blood at 5% hematocrit, in standard culturing media, supplemented with 10% human serum (Karolinska Hospital blood bank, Stockholm, Sweden), and using standard culturing techniques. Cultures were gassed with 90% NO2, 5% O2, and 5% CO2 and maintained in a shaking incubator, a technique that has been shown to effectively reduce the number of multiple-infected red blood cells. Parasites were synchronized over three consecutive rounds, using 5% sorbitol (w/v) to select for early rings and subsequently MACS sorting of the late trophozoite and schizont stages was carried out. For the purpose of generating populations of pure gametocyte cultures for validation of the putative gametocyte markers, we induced asexually replicating 3D7-164_tdT parasites and performed sexual stage induction followed by treatment with N-acetyl glucosamine for 5 days.; Parasites were cultivated in type O blood at 5% hematocrit, in standard culturing media, supplemented with 10% human serum (Karolinska Hospital blood bank, Stockholm, Sweden), and using standard culturing techniques. Cultures were gassed with 90% NO2, 5% O2, and 5% CO2 and maintained in a shaking incubator, a technique that has been shown to effectively reduce the number of multiple-infected red blood cells. Parasites were synchronized over three consecutive rounds, using 5% sorbitol (w/v) to select for early rings and subsequently MACS sorting of the late trophozoite and schizont stages was carried out. For the purpose of generating populations of pure gametocyte cultures for validation of the putative gametocyte markers, we induced asexually replicating 3D7–164_tdT parasites and performed sexual stage induction followed by treatment with N-acetyl glucosamine for 5 days. |
| Treatment Protocol: |
Samples were collected at 10, 16, 22, 32, 38, 44 ± 2 h post-invasion (p.i). At each time point 200 μl of parasite suspension was removed and stained with MitoTracker Green fluorescent dye (Life technologies, Carlsbad, CA). A total of 10 µl packed RBCs was stained with 0.5 µl MitoTracker, in 1 ml pre-heated culture media for 15 min at 37 °C. Cells were then washed 3 times in pre-heated PBS and re-suspended in 1 ml DPBS (Gibco, prod nr 14190-144). For sorting 1 µl of stained parasite cultures was diluted in 2 ml DPBS in a petri dish. Individual iRBCs were isolated using the automated, capillary-based facs-in-a-petri cell sorter (Cell Sorter, Budapest, Hungary) or the semi-automated capillary-based NK2 Transferman (Eppendorf, Hamburg, Germany). Thus, mechanical stress on the cells was minimized prior to lysis. iRBCs were collected within a 30 min window post staining and washing, and were visually monitored to ensure exclusive capture of individual single iRBCs, iRBCs in approximately 0.4-0.5 µl 1xPBS were transferred to 200 µl thin-walled PCR tubes (Corning, NY), containing 3.5ul lysis buffer (0.6% Tween-20) containing 2U/µl recombinant RNase inhibitor, supplemented with 1 µl oligo-dT (10 μM) and 1 µl dNTP mix (10 mM). Replicates of bulk samples were prepared by taking 0.4-0.5 µl of RBC suspension to 3.5 µl lysis buffer as described above. |
| Extract Protocol: |
Individual iRBCs were isolated using the automated, capillary-based facs-in-a-petri cell sorter (Cell Sorter, Budapest, Hungary) or the semi-automated capillary-based NK2 Transferman (Eppendorf, Hamburg, Germany). Replicates of bulk samples were prepared by taking 0.4–0.5 µl of RBC suspension to 3.5 µl lysis buffer as described above. |
| Library Construction Protocol: |
We generated cDNA libraries from individual iRBCs and populations of iRBCs using the Smart-seq2 protocol, with a few modifications. |
