Rneasy qiacube kit

Total RNA was isolated from pelleted blood cells (20 µL), spleen and kidney samples (25 mg) by using QIAzol Lysis Reagent (Qiagen, Hilden, Germany), TissueLyser II (Qiagen) with 5 mm steel beads for 2 × 5 min at 25 Hz followed by chloroform addition and collection of the aqueous phase. RNeasy QIAcube Kit (Qiagen) was used for automated RNA isolation of the aqueous phase as described by manufacturer. RNA was quantified in a NanoDrop ND-100 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For cell free samples (plasma), 10 µL plasma was diluted in 130 µL PBS and QIAamp Viral RNA Mini QIAcube Kit (Qiagen) used according to the manufacturer instructions. Isolated RNA was eluted in 60 µL RNase-free water and stored at −80 °C until further use.

RNA extraction and sequencing of iPSC-derived materials was performed using manufacturer's protocols for the RNeasy QIAcube kit on a QIAcube automated system (Qiagen). RNA sequencing libraries were generated, sequenced and analysed as described in the Supplementary material, ‘Methods’ section. Briefly, the data preprocessing was done with a custom Nextflow pipeline (https://github.com/wtsi-hgi/nextflow-pipelines/blob/rna_seq_5607/pipelines/rna_seq.nf), which includes the aligner parameters. Differential gene expression was analysed using the DESeq2 package^{44 (link)} with SVA correction.^{45 (link)} An adjusted P-value threshold of 0.05 was selected to identify significant differences between wild-type and mutant samples.

Viral RNAs were extracted from frozen raspberries using the MSB method as described in Raymond et al. (2021 (link)). Briefly, 40 ml of 150 mM Bis–Tris-Propane buffer pH 8 (Sigma-Aldrich) was used to elute HuNoV from the food matrix (25 g). After elution at pH 8, the eluate was clarified by centrifugation (3500×g for 10 min) and pectinase was added. Magnetic silica fine beads (AccuNanobeads, Bionneer), ascorbic and malic acid (Sigma-Alrich) were then added to the supernatant. The pH was lowered at pH 3 with HCl to maximize virus attachment to the beads. The concentrated virus was eluted by increasing the pH to 7–9. The total RNA was extracted by using the RNeasy Qiacube kit supplemented with DNase 1 as recommended by the manufacturer (QIAGEN).