Generation of Stable STING-GFP cells using retrovirus

Amplify the coding sequence for human STING using PrimeSTAR GXL DNA polymerase (Takara Bio) according to manufacturer protocol. Primers include a XhoI restriction site at the 5’ end and a SacII restriction site at the 3’ end.

Purify the amplicon from PCR reaction mixture using a NucleoSpin Gel and PCR Cleanup kit (Macherey-Nagel) and run amplicon in an agarose gel to confirm expected size.

Digest the hSTING PCR product and pEGFP-N1 plasmid using XhoI and SacII restriction enzymes (New England BioLabs) in CutSmart buffer (New England BioLabs) according to manufacturer protocol.

Run digested products in an agarose gel to confirm expected size and purify the DNA from gel using a NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel).

Ligate the digested hSTING amplicon and linearized pEGFP-N1 backbone using T4 ligase (New England BioLabs) according to manufacturer protocol.

Transform product of ligation reaction into competent E. coli, and plate on kanamycin resistant agar plates. Incubate at 37°C for 16h 0m 0s.

Pick single bacterial colonies and expand. Grow in 5mL LB media at 37°C for 16h 0m 0s.

Purify plasmid by Mini-Prep (Qiagen) and sequence.

Digest the hSTING-EGFP-N1 and pMXs-IRES-Blasticidin Retroviral Vector backbone using XhoI and NotI-HF restriction enzymes (New England BioLabs) in CutSmart buffer (New England BioLabs) according to manufacturer protocol.

Run digested products in an agarose gel to confirm expected size and purify the DNA from gel using a NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel)

Ligate the digested hSTING-EGFP amplicon and linearized pMXs-IRES-Blasticidin Retroviral Vector backbone using T4 ligase (New England BioLabs) according to manufacturer protocol.

Transform product of ligation reaction into competent E. coli, and plate on kanamycin resistant agar plates. Incubate at 37°C for 16h 0m 0s.

Pick single bacterial colonies and expand. Grow in 5mL LB media at 37°C for 16h 0m 0s.

Purify plasmid by Mini-Prep or Maxi-Prep (Qiagen) and sequence.

Plate 5 x 10⁶ Plat-A cells (Cell Biolabs) on a 10cm plate in DMEM (-P/S).

The following day, transfect cells with 9µg of pMX-STING-GFP using Fugene HD (Promega).

At 48h 0m 0s post-transfection, plate target HeLa cells at 2.5 x 10⁵ in DMEM (-P/S) 6 well format.

At 72h 0m 0s post-transfection, collect retroviral supernatant into a falcon tube and supplement with 8μg/ml Polybrene (Millipore).

Pass supernatant through 0.22μm filter to remove cellular debris and add to target HeLa cells.

At 24h 0m 0s post-transduction, remove retroviral supernatant and replace with fresh DMEM complete.

At 48h 0m 0s post-transduction, sort HeLa cells by FACS to enrich for GFP positive cells.