Additional Materials for Production of Lentivirus

• 293T Cell Line (e.g. 293T/17, ATCC, Cat.# CRL-11268™ or 293FT, Thermo Fisher, Cat.# R70007)
• Ready-to-use Lentiviral Packaging Plasmid Mix (Cellecta, Cat.# CPCP-K2A). Libraries can be packaged into lentiviral particles with nearly any 2nd or 3rd generation HIV-based lentiviral packaging mix. Cellecta’s 2nd generation lentiviral packaging mix contains two plasmids: psPAX2 and pMD2.G, pre-mixed in a ratio optimized for production of lentivirus.
• Dulbecco’s Modified Eagle Medium (D-MEM) (1X) (Corning cellgro™, Cat.# 15-013-CV)

NOTE: ADD FRESH GLUTAMINE (1X) at the time a sealed bottle of D-MEM is opened, even if the label indicates glutamine has already been added. Glutamine in solution at 4°C has a half-life of 1-2 months, so glutamine(+) D-MEM purchased “off-the-shelf” from a supplier is to be regarded as glutamine(-). In our experience, the addition of glutamine increases titer approximately 2-fold. If D-MEM comes supplemented with stable L-Alanyl-L-Glutamine dipeptide, addition of fresh glutamine is not necessary.

• Glutamine (L-Alanyl-L-Glutamine, Dipeptide L-glutamine) (Corning glutagro™, Cat.# 25-015-CI)
• Fetal Bovine Serum (Recommended: Corning, Cat.# 35-010-CV)
• Trypsin-EDTA, 1X (Corning, Cat.# 25-052-CI)
• Tissue Culture Plates
  • 150 × 25mm (15-cm) Tissue Culture Plates (Corning, Cat.# 430599)
  • 100 × 20mm (10-cm) Tissue Culture Plates (Corning, Cat.# 430167)
• Related Tissue Culture Supplies
• Lipofectamine® Transfection Reagent (Thermo Fisher, Cat.# 18324020)
• PLUS Reagent (Thermo Fisher, Cat.# 11514015)
• DNase I, RNase-free (Epicentre-Illumina, Cat.# D9905K)
• HEPES pH 7.2-7.6, 1M solution (Corning, Cat.# 25-060-CI)
• MgCl₂, 1M solution
• Nalgene™ Rapid-Flow™ Sterile 500 ml, 0.2 μm PES filter units (Thermo Fisher Cat.# 569-0020)
• LentiFuge™ Viral Concentration Reagent (Cellecta, Cat.# LFVC1) (Optional, for concentration of virus)
• D-PBS, 1X (Corning, Cat.# 21-031-CV)

Other than the specific reagents and instruments listed above, the protocols assume the user has access to standard materials (e.g., polypropylene tubes, pipette tips), equipment (table top centrifuges, pipettes, scales), and common reagents (e.g., TE buffer, ethanol) and buffers used in a typical life science laboratory.