Generation of ATG3 KO Hela cells stably expressing HaloTag-LC3B

Transform each plasmid into Stbl3 competent cells for the propagation. Next day, pick up one colony to inoculate culture in LB medium with ampicillin.

Midi prep the cultures to purify plasmids (Qiagen).

Plate 5 x 10⁶ HEK 293T cells on a 10 cm plate in DMEM medium.

HEK293T transfection:

Add retroviral packaging plasmids (pVSV-G, pBS-CMV-gagpol) and pMRX-IP-HaloTag7-LC3, 5µg each in 1.5mL warm Opti-MEM medium.

Add 45µL of TransIT-LT1 transfection reagent (Mirus) and swirl.

Incubate at Room temperature for 0h 15m 0s.

Add 1.5mL dropwise into 10 cm HEK293T plate.

At 72 hours post-transfection, collect retroviral supernatant into a falcon tube.

Concentrate retroviral supernatant to 1mL using Lenti-X concentrator (Takara) with the manufacturer instruction.

Retroviral transduction:

Plate 1 x 10⁵ ATG3 KO HeLa cells into 12-well plate one day before.

Next day, titrate 100µL, 200µL, 400µL of concentrated retroviral solution with 8µg/mL Polybrene (Sigma) into target HeLa cells.

At 24 hours post-transduction, remove retroviral supernatant and replace with fresh DMEM complete medium with 2µg/mL puromycin (GoldBio).

Perform Puromycin selection for two weeks. Confirm Halo-LC3B positive cells by FACS sorting.