TITLE: Lentivirus preparation for neuronal transdifferentiation
AUTHORS: Ching-Chieh Chou, Judith Frydman

[DESCRIPTION]
This is a protocol for the preparation of Lentiviruses.

[STEPS]
SECTION: Preparation of culture medium and plasmids
1. 1. HEK293T medium @4C lasts for 1 month. A total of 500 mL:
435 ml DMEM (High glucose, GlutaMAX) (Thermo Fisher Scientific) 
50 ml Fetal bovine serum (FBS) (Thermo Fisher Scientific) 
5 ml Penicillin-Streptomycin (Thermo Fisher Scientific) 
5 ml HEPES (1M; Thermo Fisher Scientific)
5 ml Sodium pyruvate (100 mM; Thermo Fisher Scientific)
Sterilized by filtration through a 0.22 µm filter

2. Virus medium @4C lasts for 1 month. A total of 500 mL:
470 ml DMEM (High glucose, GlutaMAX) (ThermoFisher Scientific) 
10 ml Fetal bovine serum (FBS) (Thermo Fisher Scientific) 
5 ml Penicillin-Streptomycin (Thermo Fisher Scientific) 
5 ml MEM NEAA (100X; Thermo Fisher Scientific)
5 ml HEPES (1M; Thermo Fisher Scientific)
5 ml Sodium pyruvate (100 mM; Thermo Fisher Scientific)
0.5 ml beta-mercaptoethanol (Thermo Fisher Scientific)
Sterilized by filtration through a 0.22 µm filter

3. OptiMEM+GlutaMAX (Thermo Fisher Scientific)

4. Lipofectamine 2000 (Thermo Fisher Scientific)

5. Plasmids:
Transfer: FUW-M2rtTA (Addgene plasmid# 20342), pTet-O-Ngn2-puro (Addgene plasmid# 52047), Tet-O-FUW-Ascl1 (Addgene plasmid# 27150), Tet-O-FUW-Brn2 (Addgene plasmid# 27151), Tet-O-FUW-Myt1l (Addgene plasmid# 27152)
Packaging: psPAX2 (Addgene plasmid# 12260)
Envelope: pMD2.G (Addgene plasmid# 12259) 

6. Millex syringe 0.22 & 0.45 µm filter
SECTION: Generation of Lentiviruses
2. Seed 6x106 HEK293T cells in a 10-cm dish for each transfer plasmid and culture to reach 80% confluence next day.
SECTION: Generation of Lentiviruses
3. Prepare DNA and Lipofectamine 2000 dilutions.
5 µg Transfer plasmid
4 µg Packaging plasmid
2.5 µg Envelope plasmid
DNA diluted into 300 µL OptiMEM
23 µL Lipofectamine 2000, diluted into 300 µL OptiMEM
SECTION: Generation of Lentiviruses
4. Vortex DNA and Lipofectamine dilutions, centrifuge for 5 sec and incubate at room temperature for 5 mins.
SECTION: Generation of Lentiviruses
5. Combine DNA and Lipofectamine dilutions, vortex, centrifuge and incubate the mixture for additional 15 mins.
SECTION: Generation of Lentiviruses
6. Remove old HEK293T medium, rinse the cells with OptiMEM once and then add 5 mL OptiMEM to each dish.
SECTION: Generation of Lentiviruses
7. Add a total of 600 µL of Lipofectamine/DNA mixture to the cells dropwise across the whole dish, gently shake in vertical × horizontal direction a few times and return the dishes to the incubator.
SECTION: Generation of Lentiviruses
8. After 6-hr incubation, remove the transfection medium and add 9 mL of fresh Virus medium to each dish.
SECTION: Generation of Lentiviruses
9. For additional 24-hr incubation, collect the viral particle-containing medium from each dish and store at 4°C.
SECTION: Generation of Lentiviruses
10. After medium collection, add fresh 8 mL of pre-warm Virus medium to each dish.
SECTION: Generation of Lentiviruses
11. After 24-hr incubation, collect the medium and pool it with the first collection.
SECTION: Generation of Lentiviruses
12. The cells can now be bleached and disposed of accordingly.
SECTION: Generation of Lentiviruses
13. Seal the pooled medium with parafilm and centrifuge in the swinging bucket at 400xG for 5 mins at 4°C to pellet cell debris.
SECTION: Generation of Lentiviruses
14. Carefully take the cleared supernatant and pass through a 0.45 µm filter.
SECTION: Generation of Lentiviruses
15. Viral supernatant is stored at 4°C until use and good for 2 weeks.