Qubit 1x dsdna hs assay kit

The whole genome of SARS-CoV-2 from the clinical samples was sequenced using the Oxford Nanopore sequencing technology (Oxford Nanopore Technologies, Cambridge, United Kingdom) following the ARTIC network protocol. The SARS-CoV-2 positive RNA extracts were reverse-transcripted with LunaScript^™ RT SuperMix Kit (New England Biolabs, Ipswich, United States). Multiplex PCR with ARTIC Network V3 primer pools, tilling the complete SARS-CoV-2 genome, was performed on cDNA using Q5 Hot Start High-Fidelity DNA polymerase (New England Biolabs, Ipswich, United States). The amplicons were cleaned up with AMPure XP beads (Beckman Coulter Diagnostics, California, United States), and libraries were prepared using the ligation sequencing kit (SQK-LSK109) from Oxford Nanopore Technologies (Oxford, United Kingdom). Then, libraries were quantified using QUBIT 1X dsDNA HS Assay Kit (Invitrogen, Waltham, United States), and 15 ng of each prepared library was loaded into Oxford Nanopore MinION SpotON Flow Cells FLO-MIN106D, R9.4.1 (Oxford Nanopore Technologies, Oxford, United Kingdom (Bull et al., 2020 (link); Pater et al., 2021 (link)). The FastQ files generated by the Mk1C device were used for analysis following the ARTIC Network analysis workflow and EP2ME-lab.

TCR libraries were constructed and next-generation sequencing (NSG) was performed by Chengdu ExAb Biotechnology Co, Ltd as previous report (19 (link)). Briefly, the genomic DNA was extracted using the QIAGEN OneStep reverse transcription polymerase chain reaction (RT-PCR) Kit (QIAGEN), following the manufacturer’s instructions. The PCR products were purified by using Agencourt AMPure XP beads (Beckman). Extracted genomic DNA was accurately quantified using a Qubit 1X dsDNA HS Assay Kit (Invitrogen). The TCRβ complementarity-determining region 3 (CDR3) regions were amplified by Multiplex PCR and sequenced on an Ion GeneStudio S5 System (Thermo Fisher Scientific). The TCRβ CDR3 regions were discerned based on the definition established by the International ImMunoGeneTics (IMGT) Collaboration, and the V, D, J segments contributing to each CDR3 region were identified by a standard algorithm. For each sample, we used the Shannon-Weaver index (20 (link)) and the diversity 50 (D₅₀) value to estimate the TCR diversity. The Shannon-Weaver index reflected the diversity of the TCRs. The D₅₀ index was used as a measure of the diversity and clonality of the TCRβ repertoire and defined as the proportion of TCRβ CDR3 clonetypes that account for the cumulative 50% of the total TCRβ sequences in the sample (21 (link)).

A pCS2+ plasmid containing flag-tagged mgfp5 cloned into the EcoRI/XhoI sites (a gift from Lea Starita) was transformed into two strains of competent E. coli, ER2796 (from NEB, see “Preparation of calcium competent ER2796 E. coli”) and NEB 5-alpha (NEB cat. no. C2987I), using a standard heat shock method. 50 μL of the chemically competent cells were thawed on ice and mixed with 50 ng of plasmid DNA. The mixture was placed on ice for 30 minutes before being heat shocked at 42°C for 30 seconds. After heat shock, the cells were immediately placed back on ice for 5 minutes. Following incubation, 950 μL of room temperature SOC media (NEB cat. no. B9020S) was added, and the cells were allowed to outgrow for 1 hour in the absence of selection. 50 μL of the outgrown cells were diluted in 5 mL of selective media and grown overnight with shaking at 37°C and 220 rpm. Specifically, the NEB 5-alpha cells were grown in LB media with 100 μg/mL ampicillin, while the ER2796 cells were grown in LB media with 50 μg/mL kanamycin + 100 μg/mL ampicillin. The following day, plasmid DNA was extracted from the bacterial cells using the Monarch Plasmid Miniprep Kit (NEB cat. no. T1010L) following the manufacturer’s protocol. The elution of plasmid DNA was done with sterile water and the concentration was measured using the Qubit 1X dsDNA HS Assay Kit (Invitrogen cat. no. Q33231).