NextSeq 550 paired end sequencing; scRNA-seq of rodent malaria parasites (P. berghei) and their host cells in spleen, bone marrow and blood
Blood, spleen and bone marrow was harvested from a naïve mouse or from an infected mouse 3 dpi with 1 x 106 PbmCherry parasites. Blood was collected from mice under terminal anaesthesia by cardiac puncture using a heparinized syringe. For organ collection, mice were subsequently culled by cervical dislocation, spleen, femur and tibia harvested, and the organs placed into 1 ml FACS buffer (0.2 - 0.5 % BSA/PBS, sterile filtered) on ice. Bone marrow was released by removing the ends of the bones with sharp scissors and perfusing the bones with 1 ml cold FACS buffer. Spleen and bone marrow were dissociated by gently straining the tissues through a 40 µm cell strainer using the plunger of 5 ml syringe into 50 ml tubes. 50-100 µl blood were filtered through a 40 µm cell strainer into 50 ml tubes. Cell strainers were flushed with 25 ml FACS buffer and cells were pelleted by centrifugation for 10 min at 4 °C. Blood was resuspended in 20 ml, spleen in 10 ml and bone marrow in 1 ml cold FACS buffer. Cells were counted in a Neubaur counting chamber.  Dissociated spleen, bone marrow and blood cells were strained a second time through a 40 µM filter and sorted based on mCherry fluorescence using a BD FACSAria IIu or BD FACSAria III, collecting both mCherry-positive and mCherry-negative cells. Per organ, 0.5 x 106 cells from each mCherry-positive and mCherry-negative fraction were pooled, pelleted for 10 min at 500 x g, 4 °C and resuspended in 100 µl 0.2 % BSA/PBS. Unspecific antibody binding was blocked by adding 10 µl TruStain fcX™ (anti-mouse CD16/32) (Biolegend, Cat#101320) to each sample and incubating for 10 min on ice. 20 µl cells were removed for flow analysis and remaining cells were incubated for 30 min on ice with 1 µg each of TotalSeq™-A0441 anti-mouse CD71 Antibody (Biolegend, Cat# 113824) and TotalSeq™-A0073 anti-mouse/human CD44 Antibody (Biolegend, Cat# 103045). Cells were washed by adding 1 ml 0.2 % BSA/PBS and pelleted for 5 min at 2800 x g, 4 °C. Pellets were resuspended in 1 ml 0.2 % BSA/PBS and washed twice more. Cells were filtered through a 40 µM strainer (Flowmi) and counted on an automatic cell counter (Countess 3, Invitrogen) immediately before loading onto the 10X Chromium machine.  Two Mice were infected with 10^6 mixed blood stages of PbANKA-mCherry, a line that expresses constitutively a mCherry reporter (driven by hsp70 promoter). A third mice remained naive as control.  Cells were loaded on a 10X Chromium controller and single cells prepared by droplet generation using standard protocols and V3 chemistry. RNA was released by lysis and cDNA was prepared all following standard procedures by 10X Genomics.  10x libraries were constructed following the standard 10X protocol. Three or four i7 sample indexes were used per sample.