Extract all reagent

PHHs were prepared, grown in William’s E medium, and infected with cell culture-adapted HCV as described previously [24 (link)]. The human hepatocyte cell line Huh7.5 was grown in DMEM (Life technologies) and supplemented with 10% fetal bovine serum (FBS; Thermo Scientific) and 1% penicillin-streptomycin (Life technologies). Twenty thousand cells per square centimeter were infected with HCV JFH1 [27 (link)] at an MOI of 0.1 (proliferative cells) or 0.05 (differentiated cells). In order to induce differentiation, 2% DMSO (Sigma) was added to the medium. Netrin-1-Fc (125 ng/mL) was obtained from Apotech Corp./Axxora. For Netrin-1 mRNA stability assays, cells were treated with either DRB (25 μg/mL, Sigma-Aldrich) or actinomycin D (5 μg/mL, Sigma-Aldrich) in order to inhibit transcription for the various durations of time prior to total RNA isolation using the Extract-all reagent (Eurobio).

Total RNA was extracted using Extract-All reagent (Eurobio, Les Ulis, France), according to the manufacturer’s instructions. Total RNA was digested with DNase I (Roche Diagnostics, Meylan, France) and quantified by Nanodrop at 260 nm wavelength.

Fish sampling was spread over two days during the same time period (9:00–11:00 a.m.) to avoid circadian variations. Fish from both groups were sampled each day to avoid the overlapping of group and day factors. Fish were fasted for 24 h before sampling. Fish were anesthetized (20 mg L-1), and then euthanized with a lethal dose (200 mg L-1) of tricaine methane sulfonate 222 (MS222, Pharmaq, Fordingbridge, Hampshire, UK). Full livers were collected from 15 individuals per group (5 fish per triplicate), weighed then flash frozen in liquid nitrogen and preserved at -80 °C. Total RNA was extracted from 7 individual powdered livers (ground under liquid nitrogen) per group (2 or 3 fish per triplicate). RNA extraction was performed using Extract-All reagent (Eurobio, Courtaboeuf, Essonne, France) combined with Nucleospin RNA column according to the manufacturer’s instructions (Macherey–Nagel, Düren, Germany). RNA concentration and purity were verified (260/280 ratio > 2) using an ND-1000 NanoDrop® spectrophotometer (Thermo Scientific Inc., Waltham, MA, USA) and the integrity of RNA was verified using an Agilent Bioanalyzer 2100 (Agilent Technologies Inc., Santa Clara, CA, USA). All samples showed an RNA integrity (RIN) score > 8. RNA samples were stored at − 80 °C for further RNA sequencing.

The pig jejunal explants were homogenized using 2 mL plastic bead tubes (MT Biomedicals, Illkirch, France) in 1 mL of Extract-All reagent (Eurobio, Courtaboeuf, France) in a Precellys Evolution tissue homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). ARN isolation and RT-qPCR were performed as described elsewhere [70 (link),71 (link)]. Data analysis was carried out using LinRegPCR freeware [72 (link)], and normalized against two reference genes, TATA-Box Binding Protein (TBP) and Hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1). Primers are described in Supplementary Table S2.

Jejunal explants were lysed in 1 mL of Extract All reagent (Eurobio, Les Ulis, France) with ceramic beads (Bertin Technologies, St. Quentin en Yvelines, France). Total RNA was extracted according to the manufacturer’s recommendations as previously described^{63 (link), 64 (link)}. The RNA concentration was determined by measuring the optical density at 260 nm (OD260), and the RNA integrity was assessed using both NanoDrop spectrophotometric analysis (Nanodrop ND1000, Labtech International, Paris, France) and Agilent capillary electrophoresis (Agilent 2100 Bioanalyzer, Agilent Technologies Inc., Santa Clara, CA, USA). The mean ( ± SD) RNA Integrity Number (RIN) of these mRNA preparations was 6.85 ± 0.8.

Total RNA was extracted using the Nucleospin RNA/protein kit (Macherey-Nagel) for biopsies and the Extract-all reagent (Eurobio) for cultured cells. One μg of RNA was DNAse I-digested (Promega) and reverse transcribed with 5% DMSO, using the MMLV enzyme, according to the manufacturer’s instructions (Invitrogen). Real-time quantitative RT-PCR was performed on a LightCycler 480 device (Roche) using the iQ™ SYBR^®Green Supermix (BIO-RAD). DMSO (10%, Sigma-Aldrich) was added to the PCR reaction for Netrin-1 quantification. All PCR primer sequences and qPCR conditions are reported in the S2 Table.