Illumina HiSeq 2500 sequencing; RNA-seq analysis of astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape
RNA-seq analysis of astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape
RNA was isolated from organoid tissues using the mirVana RNA isolation kit and following the Total RNA isolation protocol (Thermo-Fisher Scientific, Waltham MA). D Enteroids were derived as previously described [71–73], suspended in Matrigel (Corning) and maintained in complete L-WRN medium [74]. The Complete L-WRN medium contained 50% WRN conditioned media (ATCC), Advanced DMEM/F-12 (Gibco), 2mM GlutaMax (Invitrogen), 10mM HEPES (Invitrogen), 1% penicillin/streptomycin (Gibco), 1X N-2 media supplement (Invitrogen), 1X B-27 supplement minus vitamin A (Invitrogen), 100 μg/ml Primocin (InvivoGen), 1 mM N-Acetyl-L-cysteine (Sigma-Aldrich), 50 ng/ml EGF (Invitrogen), 10 μM SB202190 (Sigma-Aldrich), 500 nM A83-01 (Tocris), 10 mM nicotinamide (Sigma-Aldrich), 10 nM [Leu15]-Gastrin I (Sigma-Aldrich) and 10μM Y27632 (R&D Systems). For 2D monolayers, HIE were dissociated into a small cell aggregates and seeded in transwells or 48 well plates coated with human collagen type IV as described [15]. All infections were performed on 2D monolayers. HAstV-1 and HAstV-VA1 were expanded in Caco-2 cells, while HAstV-MLB1 was expanded in Huh7.5 cells to create a virus stock Briefly, 1x105 HIE were seeded in 48-well plates as 2D monolayers and incubated overnight at 37°C with 5% CO2 in complete L-WRN medium. Undifferentiated HIE were maintained in complete medium and allowed to form a confluent 2D cell monolayer, while differentiated HIE were cultured for 6 days in differentiation medium (Complete L-WRN media without WRN, nicotinamide, SB and 5% Noggin). Next, HIE were inoculated with AstV for 1 hour at 37°C and washed once with culture medium without growth factors. Fresh complete L-WRN medium was then added. Samples for 0 dpi were harvested and frozen immediately. All remaining samples were incubated at 37°C with 5% CO2 and harvested at the indicated time point. For HAstV-1 and filtered AstV-positive clinical stool sample infection, virus was treated with 10 μg/ml porcine trypsin (Sigma-Aldrich) for 30 min at 37°C before inoculating HIE for 1 hour in media containing 5 μg/ml trypsin. VA1 and MLB1 infections required no trypsin treatment. All infections were performed at MOI of 1 based on genome copies per cell.  RNA was isolated using the mirVana RNA isolation kit and following the Total RNA isolation protocol (Thermo-Fisher Scientific, Waltham MA). RNA library preparation and RNA-sequencing (single-end, 50 bp read length) were performed by the University of Michigan DNA Sequencing Core using the Illumina Hi-Seq 2500 platform.  RNA was isolated using the mirVana RNA isolation kit and following the Total RNA isolation protocol (Thermo-Fisher Scientific, Waltham MA). RNA library preparation and RNA-sequencing (single-end, 50 bp read length) were performed by the University of Michigan DNA Sequencing Core using the Illumina Hi-Seq 2500 platform.