biostudies-arrayexpressUnknownTranscriptomicsGenomicsProteomicsRoss LaidlawIllumina NovaSeq 6000RNA-seq of coding RNATrypanosoma cruziTrypanosoma cruzihttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14400Single cell RNA sequencing data across six samples (2 replicates each) of the Trypanosoma cruzi in vitro lifecycle. The fastq files named Trypomastigote3 and Trypomastigote4 are technical replicates and correspond to Trypomast 2-1 and Trypomast 2-2 respectively. Combined they make up the second biolgoycal replicate of the Trypomastigote samples.biostudies-arrayexpressLibrary Construction - Two biological replicates for each life cycle stage were prepared and sent to Novogene Co., Ltd (Cambridge, UK) for rRNA removal, library construction and sequencing. RNA quantity and integrity were measured with an Agilent 2100 bioanalyzer using the RNA Nano 6000 Assay Kit (Agilent Technologies, Santa Clara, CA, USA) and by agarose gel electrophoresis. A total of 1 µg total RNA per sample was used as input material for the lncRNA library preparation. Ribosomal RNA was depleted from total RNA and strand-specific libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (New England BioLabs, Ipswich, MA, USA) following manufacturer’s recommendations. Index codes were added to attribute sequences to each sample.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.Nucleic Acid Extraction - Total RNA was extracted using RNeasy Mini Kit (Qiagen, Hilder, Germany) following the manufacturer's protocol, with an on-column DNase digestion step with the RNAse-Free DNase Set (Qiagen).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.Sequencing - Two biological replicates for each life cycle stage were prepared and sent to Novogene Co., Ltd (Cambridge, UK) for rRNA removal, library construction and sequencing. RNA quantity and integrity were measured with an Agilent 2100 bioanalyzer using the RNA Nano 6000 Assay Kit (Agilent Technologies, Santa Clara, CA, USA) and by agarose gel electrophoresis. A total of 1 µg total RNA per sample was used as input material for the lncRNA library preparation. Ribosomal RNA was depleted from total RNA and strand-specific libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (New England BioLabs, Ipswich, MA, USA) following manufacturer’s recommendations. Index codes were added to attribute sequences to each sample.Sample Collection - Amastigotes (AMA) were obtained from Vero cells infected with ADT and washed at 6 hpi as indicated above. 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.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-SanchezfalseBulk RNA sequencing data of the in vitro lifecycle of the kinetoplastid parasite Trypanosoma cruziSingle cell RNA sequencing data across six samples (2 replicates each) of the Trypanosoma cruzi in vitro lifecycle. The fastq files named Trypomastigote3 and Trypomastigote4 are technical replicates and correspond to Trypomast 2-1 and Trypomast 2-2 respectively. Combined they make up the second biolgoycal replicate of the Trypomastigote samples.2025-08-27T00:00:00Z2024-09-02T13:49:26.137Z2024-09-02T13:49:26.137ZE-MTAB-14400ERP163727E-MTAB-14406EFO_0002944EFO_0004170EFO_0005518EFO_0003738EFO_0004184