Pmxs mir gfp puro retroviral expression vector

pMXs-miR-GFP/puro retroviral expression vector was purchased from Cell Biolabs, Inc. To generate pMXs-miR-150-GFP, miR-150 was amplified by RT-PCR from splenocyte using primers as below: miR-150 forward primer containing XhoI site, 5′-TCG ACT CGA GAC AGG AAC CCC CTC CCT CAG C-3′; miR-150 reverse primer with XhoI site, 5′-TCG ACT CGA GGG AAG GGA CCC AAG GCA TCC C-3′. The amplified PCR product was cloned into pMXs-miR-GFP/puro after treatment with XhoI restriction enzyme. Retroviruses were generated using TransIT transfection reagents and Platinum-E retroviral packing cell line as manufacturer’ protocol (Mirus Bio Corp., Madison, WI). To add-back miR-150 to CD8⁺ T cells, 1 × 10⁶ CD8⁺ T cells were transduced with multiplicity of infection of 10 of retro-miR-150-GFP or retro-miR-GFP with polybrene (8 μg/mL, Sigma-Aldrich) and IL-2 (10 U/mL, R&D systems). To measure the expression levels of anergy related genes after miR-150 add-back, CD8⁺ T cells were harvested at 2 d after virus infection and GFP-positive and negative populations were sorted using a FACS Aria cell sorter (BD Biosciences) and performed qPCR. For measurement of granzyme B, cyclin B1 and Blimp1, sorted GFP-positive and negative populations were incubated with anti-CD3/anti-CD28 (Life Technology) for 4 h, and PCR performed.

DNA sequence of precursor miR-494 was inserted between XhoI cloning sites of pMXs-miR-GFP/Puro retroviral expression vector according to the manufacturer's datasheet (Cell Biolabs, San Diego, USA). Primers and PCR conditions are reported in Table S2. Viral infection of Huh-7 cells was performed as previously described^{7 (link)}.