title: Bulk RNA sequencing data of the in vitro lifecycle of the kinetoplastid parasite Trypanosoma cruzi
design: Bulk 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 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.