Phasemaker tube

RNA isolation was carried out using TRIzol Reagent and Phasemaker tubes (Thermo Fisher Scientific) according to the manufacturer’s specifications. One hundred nanograms of RNA was converted to cDNA using the SuperScript IV one-step RT-PCR system (Invitrogen) according to the manufacturer’s specifications. Gene expression was analysed using TaqMan probes (Thermo Fisher Scientific); the supplementary methods detail the probes and cycle parameters used. Data were expressed as relative expression compared to the housekeeping genes, ACTB and HPRT1.

RNA was extracted from 100 mg of ground leaves following the protocol described in [77 (link)] with certain modifications. We used TRIzol (Thermo cat. 15596026) instead of phenol and Phasemaker tubes were used to separate the aqueous phase (Thermo cat. A33248). 10 µg of total RNA were treated with TURBO DNase I (Thermo cat. AM2238) and cDNA was synthetized using 1 µg RNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo cat. 4368814). PowerUP SYBR Green Master Mix (Thermo cat. A25741) was used for RT-qPCR using three technical replicates per reaction in a CFX96 Touch Real-Time PCR Detection System (Biorad). Primers are described in Additional file 5: Table S5. SAND and EF1a genes were used as reference for Arabidopsis and Nicotiana, respectively [78 (link)]. For experiments in Nicotiana we identified the closest orthologs of Arabidopsis genes using their protein sequence as input in the solgenomics BLAST tool against the Nicotiana benthamiana genome [79 (link)]. We used the sequence of Niben101Scf00107g03008.1 (PR1A) and Niben101scf01817g00015.1 (NbAGP41).

Total RNA was extracted from the previously homogenized frozen kidneys using TRI Reagent solution and Phasemaker tubes (A33250, Thermo Fisher Scientific, Waltham, MA, USA), followed by DNase I treatment (K2981, Thermo Fisher Scientific, Waltham, MA, USA) to eliminate genomic DNA contamination. The cDNA was synthetized from 1.5 µg of total RNA using a high-capacity cDNA reverse transcription kit (4368814, Applied Biosystems, Waltham, MA, USA). According to the measurement of the relative mRNA levels, quantitative (q) RT-PCR was performed using SYBR Select Master Mix for CFX (4472942, Applied Biosystems, Waltham, MA, USA) or TaqMan Gene Expression Master Mix (4369514, Applied Biosystems, Waltham, MA, USA). Each mRNA from a single sample was measured in duplicate, using M36b4 and B2m as housekeeping genes. The sequences of the primers used in the qPCR are presented in Supplemental Data (Table S1). Results were obtained by the relative standard curve method and expressed as fold increases, using the chow-diet experimental group as the reference.

Hundred ml each of NS and BAL fluid collected on day 2, 4 and 6 pc and rectal swabs collected on day 6 pc were inactivated in a BSL3 laboratory using 400 ml buffer AVL (Qiagen) and 500 ml ethanol, and RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen) according to the manufacturer’s protocol. To extract total RNA from lung homogenates harvested on day 6 pc, 300 ml of each lung homogenate (at a concentration of 0.1 g of tissue/ml) was mixed with 900 ml TRIzol LS (Thermo Fisher) using Phasemaker Tubes (Thermo Fisher) and RNA was extracted using the PureLink RNA Mini Kit (Thermo Fisher) following the manufacturer’s instructions. Then, the SARS-CoV-2 genomic N RNA and subgenomic E mRNA were quantified in triplicate using the TaqMan RNA-to-Ct 1-Step Kit (Thermo Fisher) using previously reported TaqMan primers/probes (Chandrashekar et al., 2020 (link); Corman et al., 2020 ; Wolfel et al., 2020 (link)) on the QuantStudio 7 Pro (ThermoFisher). Standard curves were generated using serially diluted pcDNA3.1 plasmids encoding gN, gE, or sgE sequences. The limit of detection was 2.57 log₁₀ copies per ml of NS, BAL fluid, or rectal swabs and 3.32 log₁₀ copies per g of lung tissue.

TCR libraries of MHC-tetramer+ and MHC-tetramer- fractions were prepared as described previously^{65 (link)}. RNA was isolated from Trizol reagent (Thermo Fisher Scientific) using Phasemaker Tubes (Thermo Fisher Scientific), the cDNA synthesis reaction for TCR β chains was carried out with a primer to the C-terminal region and SMART-Mk, providing a 5′ template-switch effect and containing a sample barcode for contamination control as well as a unique molecular identifier. TCR libraries of total PBMC samples were generated using the human multiplex TCR Kit (MiLaboratories) according to the manufacturer’s instructions. Sequencing was performed with an Illumina MiSeq or NextSeq platform. TCR repertoire data were analyzed using MIXCR^{66 (link)}, MIGEC^{67 (link)}, and VDJtools software^{68 (link)} with default settings.

Hamsters were euthanized at the indicated timepoints following infection and lung weights were recorded. Lungs were lysed in Trizol (ThermoFisher) and dissociated in gentle MACS M tubes using the gentleMACS Octo Dissociator (Miltenyi Biotec). Samples were transferred into Phasemaker tubes (ThermoFisher) and chloroform was added (200 μl chloroform/1 ml Trizol). After vigorous shake, tubes were rested for 5 min and then centrifuged for 15 min at 12,000g at 4°C. The aqueous phase containing the RNA was transferred into a new tube and RNA extraction was performed by using RNeasy mini kit (Qiagen). SARS-CoV-2 lung titers were determined by qRT-PCR assay using TaqMan Fast Virus 1-Step Master Mix and specifically designed primers and a TaqMan probe that bind a conserved region in the nucleocapsid gene of SARS-CoV-2 (2019-nCoV_N1-F :5’- GAC CCC AAA ATC AGC GAA AT-3’. 2019-nCoV_N1-R: 5’- TCT GGT TAC TGC CAG TTG AAT CTG-3’. 2019-nCoV_N1-P: 5’- FAM-ACC CCG CAT TAC GTT TGG TGG ACC-BHQ1-BHQ1–3’). qPCR was performed using an Applied Biosystems QuantStudio 6 Flex cycler using the following parameters: 50°C for 5 min, 95°C for 20 s followed by 40 cycles of 95°C for 3 s, and 30 s at 60°C. Signal from unknown samples was compared to a known standard curve (obtained through BEI Resources, NIAID, NR-52358) and viral titers were expressed as RNA copies/mg tissue.

Following homogenization of the two lungs and turbinates in our BSL-3 facility, RNA was extracted with Trizol Plus RNA Purification Kit with Phasemaker tubes (Thermo Fisher)¹² . The procedure was shown by us to eliminate detectable infectious virus and the RNA was approved for transfer to a BSL-2 facility where contaminating DNA was removed using theTurbo DNA-free kit (Thermo Fisher) and RNA was reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). CoV-2 S and N transcripts and 18S rRNA were quantified by ddPCR with specific primers (CoV-2 RNA Leader, Forward—CGA TCT CTT GTA GAT CTG TTC TCT AAA C; CoV-2 S, Reverse—TCT TAG TAC CAT TGG TCC CAG AGA; CoV-2 N, Reverse—GGT CTT CCT TGC CAT GTT GAG T; 18 S, Forward—GGC CCT GTA ATT GGA ATG AGT C; 18 S, Reverse—CCA AGA TCC AAC TAC GAG CTT)¹² using an automated droplet generator and QX200 Droplet Reader (Bio-Rad). The values for CoV-2 S and CoV-2 N sgRNAs were determined relative to the 18S RNA in the same sample.