Materials and Reagents

1. The Huh-7.5.1 cell line (kindly provided by Dr. Francis Chisari from the Scripps Research Institute, USA)
   
2. Dulbecco's modified Eagle medium (DMEM) (Corning, Cellgro^®, catalog number: 10-017-CV )
   
3. Fetal bovine serum (FBS) (Omega Scientific, catalog number: FB-11 )
   
4. 100x non-essential amino acids solution (Life Technologies, catalog number: 11140050 )
   
5. 1 M HEPES (Life Technologies, catalog number: 15630080 )
   
6. 100x Penicillin-Streptomycin-Glutamine (Life Technologies, catalog number: 10378016 )
   
7. 10x trypsin supplemented with EDTA (Life Technologies, Gibco^®, catalog number: 15400054 )
   
8. Plasmid that carries the HCV viral genome (pFNX-HCV) was synthesized based on the chimeric sequence of J6/JFH1 virus
   Note: In this protocol, we are taking the HCV NS5A mutant library as an example to describe the procedures to relative fitness determination (Qi et al., 2014). A mutant virus library where each codon of interest was individually substituted with ‘NNK’, where N represents random incorporation of A/T/G/C; K represents random incorporation of T/G. The randomized codons therefore include 32 nucleotide combinations, which cover all possible amino acid.
   
9. 100% ethanol (Decon Labs, catalog number: 2701 )
   
10. QIAamp Viral RNA Mini Kit for viral RNA purification (QIAGEN, catalog number: 52906 )
    
11. Sterile, RNase-free pipet tips (with aerosol barriers for preventing cross-contamination) (OLYMPUS, catalog numbers: 24-401 , 24-404 , 24-412 , 24-430 )
    
12. SuperScript^TM III Reverse Transcriptase Kit (Life Technologies, Invitrogen^TM, catalog number: 18080-044 )
    
13. RNaseOUT Recombinant Ribonuclease Inhibitor (Life Technologies, Invitrogen^TM, catalog number: 10777-019 )
    
14. KOD Hot Start DNA Polymerase Kit (Novagen^®, catalog number: 71086-4 )
    
15. PureLink^® Quick PCR Purification Kit (Life Technologies, Invitrogen^TM, catalog number: K3100-02 )
    
16. T4 Polynucleotide Kinase (PNK) (New England Biolabs, catalog number: M0201S )
    
17. NEB buffer 2 (New England Biolabs, catalog number: B7002S )
    
18. dATP (100 mM) (Life Technologies, Invitrogen^TM, catalog number: 10216-018 )
    
19. Klenow Fragment (3’ to 5’ exo-) enzyme (New England Biolabs, catalog number: M0212S )
    
20. T4 DNA Ligase Kit (Life Technologies, Invitrogen^TM, catalog number: 15224-017 )
    

Equipment

1. 15 cm cell culture dishes (Genesee Scientific, catalog number: 25-203 )
   
2. T-150 cell culture flasks (Genesee Scientific, catalog number: 25-211 )
   
3. 37 °C, 5% CO₂ cell culture incubator
   
4. 1.7 ml Microtubes (1.5 ml) (Genesee Scientific, catalog number: 22-282 )
   
5. Falcon 50 ml tubes (Corning, catalog number: 14-432-22 )
   
6. Falcon 15 ml tubes (Corning, catalog number: 05-527-90)
   
7. Microcentrifuge (with rotor for 1.5 ml and 2 ml tubes) (Eppendorf, model: 5424 )
   
8. Centrifuge (with rotor for 15 ml and 50 ml Falcon tubes) (Thermo Fisher Scientific, Legend RT)
   
9. NanoDrop ND-1000 UV Spectrophotometer (Thermo Fisher Scientific)
   
10. Thermal cycler (Eppendorf, catalog number: 950030050 )
    

Procedure