RNA-seq analysis of HAstV1, HAstV4, MLB1, MLB2 and VA1 astrovirus infection in cell culture
RNA-seq analysis of HAstV1, HAstV4, MLB1, MLB2 and VA1 astrovirus infection in cell culture
Cells were harvested at 6, 12, 18 or 24 h as indicated.  Caco-2 (ATCC) and Huh7.5.1 cells (Apath, Brooklyn, NY) were maintained at 37 °C in DMEM supplemented with 5% fetal bovine serum (FBS), 1 mM L-glutamine, 10% FBS, non-essential amino acids and antibiotics. Caco-2 cells were infected at MOI 5 with HAstV1, HAstV4 or VA1. Huh7.5.1 cells were infected at MOI 5 with MLB1 or MLB2.  Total RNA was extracted by Direct-zol RNA Prep kit (Zymo research). RNA was fragmented, and 50-150 nt fragments were used for RNA-seq library preparation using an Illumina library protocol as described in Lulla et al (2019, PMID 30478287) and Irigoyen et al (2016, PMID 26919232), except that the Ribo-Zero rRNA removal kit (Illumina) was used to deplete ribosomal RNA. For the indicated experiments, proteinase K (NEB) treatment was performed using 10 µg of total RNA extracted from infected cells, 1% SDS (final concentration) at 42°C for 45 minutes.  Total RNA was extracted by Direct-zol RNA Prep kit (Zymo research). RNA was fragmented, and 50-150 nt fragments were used for RNA-seq library preparation using an Illumina library protocol as described in Lulla et al (2019, PMID 30478287) and Irigoyen et al (2016, PMID 26919232), except that the Ribo-Zero rRNA removal kit (Illumina) was used to deplete ribosomal RNA. For the indicated experiments, proteinase K (NEB) treatment was performed using 10 µg of total RNA extracted from infected cells, 1% SDS (final concentration) at 42°C for 45 minutes. 