Lentiviral vector

Viral vectors were produced by Obio Technology Corp., Ltd (Shanghai, China) including pLenti-EF1a-luc-F2A-Puro-CMV-FEN1-3FLAG and pLKD-CMV-G&PR-U6-shRNA-FEN1. We also purchased a lentiviral vector containing primary transcripts of miR-140-5p (Applied Biological Materials, Canada). Small interfering RNA (shRNA) sequences targeting human FEN1 were designed and are listed in Supplementary Table 1. Primer sequences for FEN1 overexpression vectors are listed in Supplementary Table 2. Transfections were performed according to the manufacturer's protocols. Briefly, cells grown to ~40% confluency were infected at a multiplicity of infection (MOI) of 40 using 5 μg/ml polybrene to aid viral attachment. Cells were selected with 2 μg/mL puromycin for a minimum of 4 weeks to select stably transfected lines.

Primary cells were transfected with hTERT contained within a lentiviral vector (Applied Biological Materials, Richmond, BC). The cells were maintained in 6-well culture plates and transfection was initiated at 70% confluency. Culture media was replaced with a 1:1 dilution of media and Lenti-hTERT stock solution in the presence of 5μg/mL polyprene and incubated overnight. The following day the virus containing media was aspirated and replaced with fresh culture media for 72 h. Cells were washed with PBS and passaged using Trypsin-EDTA (0.05%) (Life Technologies, Carlsbad, CA). Selection was performed by resuspending cells in fresh culture media in the presence of 4 μg/mL puromycin. Puromycin resistant pAECs were expanded into a homogenous cell line after 15 days in culture.

A lentiviral vector expressing HLA-B*35 (or HLA-B*8) was generated by Applied Biological Materials Inc (Richmond, BC, Canada). Briefly, the cDNA encoding HLAB*35/B*8 was cloned in the shuttle vector pLenti-II-HA-CMV, which contains a HIS tag driven by a separate CMV promoter, and was used to generate recombinant lentiviruses. Lentivirus pLenti-II-HA-CMV was used as a control vector.
Healthy control PBMCs were plated in six-well plates at a density of 0.8-1x10⁶ cells/well in RPMI supplemented with 10 % FCS and 1 % AA overnight prior to transduction. Transductions were performed using M.O.I.’s ranging from 0.1 to 1 mixing the appropriate volume of virus with 8 mg/ml Polybrene (Sigma, St. Louis, MO, USA), and adding the mixture to the cells together with RPMI to achieve a total volume of 500 μL per well. After 5–6 h incubation at 37 °C an additional 500 μL of complete RPMI was added, cells were centrifuged for 30 min at 1200 rpm and culture medium was aspirated and replaced by fresh RPMI. The transduced cells were collected after 72 h. Total RNA was extracted using Qiagen’s RNeasy Mini Kits according to the manufacturer’s protocol.