E-MTAB-14400:120 hpi amastigote 2
2025-08-27
ENA first public
ERC000011
ENA-CHECKLIST
University of Glasgow University of Edinburgh
INSDC center name
public
INSDC status
ERS20928036
SRA accession
E-MTAB-14400:120 hpi amastigote 2
Submitter Id
ArrayExpress
broker name
120 hpi amastigote
cell_type
not collected
collection date
Protocols: 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. 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).  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.
description
not collected
geographic location (country and/or sea)
not applicable
isolate
Trypanosoma cruzi
organism
Trypanosoma cruzi
organism
Trypanosoma cruzi
scientific_name
120 hpi amastigote 2
title