Single cell transcriptomics of trypanosoma brucei during cattle infection
Single cell transcriptomics of trypanosoma brucei during cattle infection
200 ml of blood was collected for each scRNA-seq sample on days 4, 11 and 23. To obtain a pure parasite sample, a combination of red blood cell (RBC) lysis and DE52 column purification was used. Each whole blood sample was aliquoted into eight 50 ml falcons (~16 ml per falcon) and centrifugedat 1,200 x g for 15 min without breaks to separate blood. The plasma was carefully removed, leaving RBCs and buffy coat behind where parasites will be located. A hypotonic lysis buffer (0.3 % NaCl in H2O) was added to each tube to a total volume of 45 ml and inverted. 5 ml of 10X PBS (Phosphate Buffered Saline) was then added and tubes inverted to halt RBC lysis. Samples were centrifuged at 80 x g for 20 min to pellet trypanosomes and retain as much of the blood cells in the supernatant as possible. Supernatant was then removed, and each pellet was combined by resuspending all in 10 ml of PSG (1X PBS + 1% D-glucose) and combining into one 50 ml tube. The combined sample was centrifuged at 400 x g for 10 min and supernatant removed, leaving 2 – 3ml of volume to resuspend the pellet in. The resuspension was mixed 1:1 with DE52 cellulose slurry and then applied to a glass filter column containing ~100 ml of DE52 cellulose pre-washed with warm PSG. Columns were slowly washed with ~100 ml of PSG to obtain pure trypanosomes and retain blood cells in the columns.  Eluted parasites were then centrifuged at 400 x g for 10 min to pellet. Supernatant was removed to leave ~1ml of PSG to resuspend the pellet. Parasites were then counted with a haemocytometer and adjusted to 1000 cell/μl by transfer to a clean Eppendorf, further centrifugation (400 x g for 10 min) and resuspension in an appropriate volume of PSG. Cells were counted again and adjusted as needed before 20,000 were loaded onto the Chromium (10X Genomics) controller for scRNA-seq. Chromium scRNA-seq 3' expression kit, as standard protocol.  Chromium scRNA-seq 3' expression kit, as standard protocol.