TITLE: Generation of stable ATG3-mCherry wild-type or mutans HeLa cells with HaloTag-LC3B using lentivirus
AUTHORS: Xuefeng Ren

[DESCRIPTION]
This protocol details the generation of stable ATG3-mCherry wild-type or mutant HeLa cells with HaloTag-LC3B using lentivirus.

[STEPS]
SECTION: Cloning pCDH1-CMV-ATG3-mCherry-Hygro lentiviral vector (ATG3 wild-type or mutants)
1. Amplify the coding sequence for human ATG3 (1-125), mCherry or ATG3 (126-314) fragment using Q5 High Fidelity DNA Polymerase and purify PCR products using Gel extraction kit (Bio Basic).
SECTION: Cloning pCDH1-CMV-ATG3-mCherry-Hygro lentiviral vector (ATG3 wild-type or mutants)
2. PCR products in step 1 are used as templates to run the second step PCR to amplify ATG3 (1- 125)-mCherry-ATG3(126-314) and gel extracted. Primers include a NheI restriction site at the 5’ end and a BamHI site at the 3’ end.
SECTION: Cloning pCDH1-CMV-ATG3-mCherry-Hygro lentiviral vector (ATG3 wild-type or mutants)
3. Digest the ATG3-mCherry PCR product and pCDH1-CMV-HT-LC3-SV40-Hygro plasmid using NheI and BamHI restriction enzymes (New England BioLabs).
SECTION: Cloning pCDH1-CMV-ATG3-mCherry-Hygro lentiviral vector (ATG3 wild-type or mutants)
4. Purify the digested DNAs using Gel extraction kit (Bio Basic).
SECTION: Cloning pCDH1-CMV-ATG3-mCherry-Hygro lentiviral vector (ATG3 wild-type or mutants)
5. Ligate the digested ATG3-mCherry and linearized pCDH1-CMV-hygro backbone using T4 DNA ligase (New England BioLabs) according to manufacturer instructions.
SECTION: Cloning pCDH1-CMV-ATG3-mCherry-Hygro lentiviral vector (ATG3 wild-type or mutants)
6. Transform ligation product into XL10-Gold competent E. coli and plate on Ampicillin resistant plates. Incubate the plate at 37 °C for 960 min.

SECTION: Cloning pCDH1-CMV-ATG3-mCherry-Hygro lentiviral vector (ATG3 wild-type or mutants)
7. Inoculate one single colony in 5 mL LB medium with ampicillin at 37 °C 960 min.

SECTION: Cloning pCDH1-CMV-ATG3-mCherry-Hygro lentiviral vector (ATG3 wild-type or mutants)
8. Miniprep plasmids (Bio Basic) and send for sequencing.
SECTION: HEK293T lentiviral transfection
9. Each plasmid for lentiviral transfection is transformed into Stbl3 competent cells for the propagation. Next day, pick up one colony to inoculate overnight culture in LB medium with ampicillin.
SECTION: HEK293T lentiviral transfection
10. Midi prep the cultures to purify plasmids (Qiagen).
SECTION: HEK293T lentiviral transfection
11. Plate 5 x 106 HEK 293T cells on a 10 cm plate in DMEM medium.
SECTION: HEK293T lentiviral transfection
12. HEK293T transfection:
SECTION: HEK293T lentiviral transfection
12.1. Add retroviral packaging plasmids (pVSV-G, pCMV R8.74) and pCDH1-CMV-ATG3-mCherryHygro (ATG3 wild-type or mutants), 5 µg each in 1.5 mL warm Opti-MEM medium.

SECTION: HEK293T lentiviral transfection
12.2. Add 45 µL of TransIT-LT1 transfection reagent (Mirus) and swirl.

SECTION: HEK293T lentiviral transfection
12.3. Incubate at Room temperature for 15 min.

SECTION: HEK293T lentiviral transfection
12.4. Add 1.5 mL dropwise into 10 cm HEK293T plate.

SECTION: HEK293T lentiviral transfection
12.5. At 72 hours post-transfection, collect retroviral supernatant into a falcon tube.
SECTION: HEK293T lentiviral transfection
13. Concentrate retroviral supernatant to 1 mL using Lenti-X concentrator (Takara) with the manufacturer instruction.

SECTION: Transduction of HeLa cells with pCDH-CMV-ATG3-mCherry lentivirus
14. Plate 1 x 105 ATG3 KO/HaloTag-LC3B HeLa cells into 12-well plate one day before.
SECTION: Transduction of HeLa cells with pCDH-CMV-ATG3-mCherry lentivirus
15. Next day, titrate 100 µL, 200 µL, 400 µL of concentrated retroviral solution with 8 µg/mL Polybrene (Sigma) into target HeLa cells.
SECTION: Transduction of HeLa cells with pCDH-CMV-ATG3-mCherry lentivirus
16. At 24 hours post-transduction, remove retroviral supernatant and replace with fresh DMEM complete medium with 0.5 mg/mL hygromycin B (Gibco).

SECTION: Transduction of HeLa cells with pCDH-CMV-ATG3-mCherry lentivirus
17. After one week of hygromycin B selection, sort HeLa cells by FACS to enrich for mCherry positive cells using Wolf G2 cell sorter (Nanocellect).