title: Single cell RNA sequencing data of the in vitro lifecycle of the kinetoplastid parasite Trypanosoma cruzi
design: Single cell RNA sequencing data of the in vitro lifecycle of the kinetoplastid parasite Trypanosoma cruzi
library_construction_protocol: Amastigotes (AMA) were obtained from Vero cells infected with ADT and washed at 16 hpi. 48 hpi, intracellular AMA were isolated from the host cells following the protocol described by Dumoulin and colleagues (Dumoulin et al. 2020), with minimum modifications. MOI 10 was used for the sample. Infected monolayers were washed with ice-cold PBS and scraped into 3 ml PBS. Infected cells suspensions were lysed using the gentleMACSTM dissociator with gentleMACSTM M tubes (Miltenyi Biotec, Bergisch Gladbach, Germany) and the “Protein” protocol. The ensuing lysates were passed through a PD-10 desalting column (Cytiva, Little Chalfont, UK) equilibrated with PBS to remove debris. AMA were eluted in 3.5 ml ice-cold PBS and were washed 3 times in 15 ml PBS by centrifugation at 1,350 x g for 5 min at 4 °C, to remove host cell RNA and debris. Single-cell libraries were generated using the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 and the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 - Dual Index (10X Genomics) for the first and second biological replicates respectively. Briefly, single-cell suspensions were loaded onto a Chromium Controller instrument in a Chromium Next GEM Chip G (10X Genomics) to obtain single‐cell Gel beads in EMulsion (GEMs). Barcoded cDNA was obtained from GEMs and amplified by PCR in a PCRmax Alpha Cycler 1 thermal cycler (Cole-Parmer, Saint Neots, UK). End repair and A‐tailing, adapter ligation, post‐ligation cleanup with SPRIselect (Beckman Coulter, Brea, CA, USA), and sample index PCR and cleanup steps were performed as per the manufacturer's instructions. Final library sample index PCR cycle parameters were selected based on quantification of barcoded cDNA samples with a Qubit 2.0 (Life technologies, Carlsbad, CA, USA), using the Qubit dsDNA HS Assay Kit (Life technologies, Eugene, OR, USA) Single-cell libraries were generated using the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 and the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 - Dual Index (10X Genomics) for the first and second biological replicates respectively. Briefly, single-cell suspensions were loaded onto a Chromium Controller instrument in a Chromium Next GEM Chip G (10X Genomics) to obtain single‐cell Gel beads in EMulsion (GEMs). Barcoded cDNA was obtained from GEMs and amplified by PCR in a PCRmax Alpha Cycler 1 thermal cycler (Cole-Parmer, Saint Neots, UK). End repair and A‐tailing, adapter ligation, post‐ligation cleanup with SPRIselect (Beckman Coulter, Brea, CA, USA), and sample index PCR and cleanup steps were performed as per the manufacturer's instructions. Final library sample index PCR cycle parameters were selected based on quantification of barcoded cDNA samples with a Qubit 2.0 (Life technologies, Carlsbad, CA, USA), using the Qubit dsDNA HS Assay Kit (Life technologies, Eugene, OR, USA)