biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsRoss LaidlawIllumina NovaSeq 6000RNA-seq of coding RNA from single cellsTrypanosoma cruziTrypanosoma cruzihttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14406Single cell RNA sequencing data across seven samples of the Trypanosoma cruzi in vitro lifecycle. The samples have the following lifecycle stage compositions: Sample 1 (fastq file prefix = cycle_epimast) - Mix of exponential growth growth epimastigotes and amastigotes derived 120 hours post infection of Vero cells with trypomastigotes. Sample 2 (fastq file prefix = cycle_amast) - Mix of amastigotes derived 6 and 24 hours post infection of Vero cells with trypomastigotes. Sample 3 (fastq file prefix = cycle_metacyc1) - Mix of stationary growth phase epimastigotes and metacyclic trypomastigotes (first of two biological replicates). Sample 4 (fastq file prefix = cycle_metacyc2) - Mix of stationary growth phase epimastigotes and metacyclic trypomastigotes (second of two biological replicates). Sample 5 (fastq file prefix = cycle_trypomast)- Trypomastigotes harvested from culture 7 days post infection of Vero cells with trypomastigotes. Sample 6 (fastq file prefix = LifeCycleMix1) - A mix of amastigotes derived 6, 24 and 120 hours hours post infection of vera cells with trypomastigotes, exponential and stationary growth phase epimastigotes, metacyclic trypomastigotes and trypomastigotes harvested from culture 7 days post infection of Vero cells with trypomastigotes (first of two biological replicates). Sample 7 (fastq file prefix = LifeCycleMix2) - A mix of amastigotes derived 6, 24 and 120 hours hours post infection of Vero cells with trypomastigotes, exponential and stationary growth phase epimastigotes, metacyclic trypomastigotes and trypomastigotes harvested from culture 7 days post infection of Vero cells with trypomastigotes (first of two biological replicates). Sample 8 (fastq file prefix = A2, B2, C2 and D2) - Amastigotes derived 48 hours post infection of Vero cellsbiostudies-arrayexpressNucleic Acid Extraction - 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)Sample Collection - Epimastigotes (EP) were collected in the exponential growth phase, from 2 days-old cultures initiated with 3x10/1^5 cells/ml. A mixture of stationary phase epimastigotes and metacyclic trypomastigotes (SE/MT) was obtained from a 10 days-old culture initiated with 3x10/1^5 epimastigotes/ml.Sample Collection - Amastigotes (AMA) were obtained from Vero cells infected with ADT and washed at 6 hpi. At the time points selected (6, 24, and 120 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 1.5 was used for 120 hpi samples and MOI 10 for 6 and 24 hpi samples. 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.Library Construction - 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)Sample Collection - 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.Sequencing - Libraries were quantified by Qubit and quality was tested in an Agilent 2100 Bioanalyzer, before sequencing on the Illumina NovaSeq 6000 platform with PE150 strategy for 20 Gb raw data per sample.Sample Collection - A mixture of stationary phase epimastigotes and metacyclic trypomastigotes (SE/MT) was obtained from a 10 days-old culture initiated with 3x10/1^5 epimastigotes/ml.Sample Collection - For the life cycle mixes equal quantities of EP, SE/MT, ADT, AMA_6, AMA_24 and AMA_120 (for 21,000 cells in total) were pooled. For this pool, two technical replicates were collected (labelled MIX1 and MIX2)Sample Collection - Amastigote derived trypomastigotes (ADT) were harvested from the supernatant of infected Vero cells at 7 dpi. EP, SE/MT and ADT were put in falcon tubes and left undisturbed for 1 h (at 28 °C for EP and SE/MT, and at 37 °C 5\\% CO2 for ADT). To avoid collecting dead parasites and co-purified Vero cells, the supernatant, containing viable and motile cells, was collected. Parasites were pelleted and washed once in ice-cold phosphate-buffered saline (PBS) by centrifugation at 1,350 x g for 5 min at 4 °C.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesManu De RyckerThomas OttoRoss LaidlawMarta Garcia-SanchezfalseSingle cell RNA sequencing data of the in vitro lifecycle of the kinetoplastid parasite Trypanosoma cruziSingle cell RNA sequencing data across seven samples of the Trypanosoma cruzi in vitro lifecycle. The samples have the following lifecycle stage compositions: Sample 1 (fastq file prefix = cycle_epimast) - Mix of exponential growth growth epimastigotes and amastigotes derived 120 hours post infection of Vero cells with trypomastigotes. Sample 2 (fastq file prefix = cycle_amast) - Mix of amastigotes derived 6 and 24 hours post infection of Vero cells with trypomastigotes. Sample 3 (fastq file prefix = cycle_metacyc1) - Mix of stationary growth phase epimastigotes and metacyclic trypomastigotes (first of two biological replicates). Sample 4 (fastq file prefix = cycle_metacyc2) - Mix of stationary growth phase epimastigotes and metacyclic trypomastigotes (second of two biological replicates). Sample 5 (fastq file prefix = cycle_trypomast)- Trypomastigotes harvested from culture 7 days post infection of Vero cells with trypomastigotes. Sample 6 (fastq file prefix = LifeCycleMix1) - A mix of amastigotes derived 6, 24 and 120 hours hours post infection of vera cells with trypomastigotes, exponential and stationary growth phase epimastigotes, metacyclic trypomastigotes and trypomastigotes harvested from culture 7 days post infection of Vero cells with trypomastigotes (first of two biological replicates). Sample 7 (fastq file prefix = LifeCycleMix2) - A mix of amastigotes derived 6, 24 and 120 hours hours post infection of Vero cells with trypomastigotes, exponential and stationary growth phase epimastigotes, metacyclic trypomastigotes and trypomastigotes harvested from culture 7 days post infection of Vero cells with trypomastigotes (first of two biological replicates). Sample 8 (fastq file prefix = A2, B2, C2 and D2) - Amastigotes derived 48 hours post infection of Vero cells2025-08-27T00:00:00Z2025-01-17T14:02:53.86Z2024-09-02T13:50:27.259ZE-MTAB-14406ERP163729EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0004184