TITLE: Lentiviral transduction of iPSCs with sgRNAs and sgRNA libraries
AUTHORS: Ruilin Tian, Jason Hong, Sydney Sattler, Martin Kampmann

[STEPS]
?. [Day 0: Seeding]
18 – 24 hours before transfection, seed 293T cells into a 6 well plate or other format with a density that will make the cells 80 – 95 % confluent on the day of transfection. Refer to a seeding chart if necessary to seed appropriate density.
?. [Day 0: Seeding]
Incubate overnight.
?. [Day 1: Transfection]
Change 293T media with fresh DMEM.
?. [Day 1: Transfection]
Warm TransIT-Lenti Reagent to .
0 Room temperature
?. [Day 1: Transfection]
Gather Opti-Mem, DNA, and packaging mix and refer the table below for the recommended amount of reagents to add based on the format of 293Ts seeded. Amounts refer to each well of a plate.  ABCDEFGH1Culture vessel48-well plate24-well plate12-well plate6-well plate10-cm dishT75 flask15-cm dish2Surface area1.0 cm^21.9cm^23.8 cm^29.6 cm^259 cm^275 cm^2145cm^23Complete growth medium263 μl0.5 ml1.0 ml2.0 ml10 ml15 ml30 ml4Opti-Mem serum-free medium26 μl50 μl100 μl200 μl1.0 ml1.5 ml3.0 ml5Transfer DNA (1 μg/μl stock)0.13 μl0.25 μl0.5 μl1.0 μl5 μl7.5 μl15 μl6Packaging DNA Premix (1 μg/μl stock)0.13 μl0.25 μl0.5 μl1.0 μl5 μl7.5 μl15 μl7TransIT-Lenti Reagent0.78 μl1.5 μl3 μl6 μl30 μl45 μl90 μl
Typically for individual sgRNAs, 2 wells of a 6 well plate per sgRNA will produce enough Lentivirus particles. For sgRNA libraries (containing up to 50,000 elements), a 15 cm dish can be used. This can be scaled down for smaller libraries.
ABCDEFGH1Culture vessel48-well plate24-well plate12-well plate6-well plate10-cm dishT75 flask15-cm dish2Surface area1.0 cm^21.9cm^23.8 cm^29.6 cm^259 cm^275 cm^2145cm^23Complete growth medium263 μl0.5 ml1.0 ml2.0 ml10 ml15 ml30 ml4Opti-Mem serum-free medium26 μl50 μl100 μl200 μl1.0 ml1.5 ml3.0 ml5Transfer DNA (1 μg/μl stock)0.13 μl0.25 μl0.5 μl1.0 μl5 μl7.5 μl15 μl6Packaging DNA Premix (1 μg/μl stock)0.13 μl0.25 μl0.5 μl1.0 μl5 μl7.5 μl15 μl7TransIT-Lenti Reagent0.78 μl1.5 μl3 μl6 μl30 μl45 μl90 μl
?. [Day 1: Transfection]
Add Opti-MEM into a sterile tube.
?. [Day 1: Transfection]
In another tube, mix Packaging DNA Premix and DNA.
?. [Day 1: Transfection]
Add the DNA mix to the Opti-MEM and mix gently.
?. [Day 1: Transfection]
Add TransIT-Lenti Reagent to the mixture and mix gently.
?. [Day 1: Transfection]
Incubate for  for transfection complexes to form.
?. [Day 1: Transfection]
Add all of the TransIT-Lenti:DNA complex mixture to the 293Ts dropwise and gently swirl to mix.
?. [Day 1: Transfection]
Incubate for 2 days. If a fluorescent marker is included in your DNA, you can check if cells are making virus by checking fluorescence after 24 hours.
?. [Day 3: Harvest]
With a 12 ml syringe, take up the media/supernatant of the cells.
?. [Day 3: Harvest]
Put a 0.45 μm filter on the syringe and filter the supernatant into a fresh 15 ml conical tube.
Change the filter if it becomes hard to push. Do not push too hard that bubbles are coming out.
?. [Day 3: Harvest]
Add 1:4 ratio of cold viral precipitation solution (e.g. 0.25 mL viral precipitation solution for 1 mL of viral supernatant).
?. [Day 3: Harvest]
Incubate the viral supernatant at  for at least  and up to 3 days but no more than 3 days.
4 °C
?. [Day 3: Harvest]
Cool down the centrifuge to .
4 °C
?. [Day 3: Harvest]
Spin down viral supernatant for  at .
Centrifuge: 1500 34
?. [Day 3: Harvest]
The pellet will contain the virus. Resuspend the pellet with  of your media of choice.
1 ml
?. [Day 3: Harvest]
Virus can be aliquoted and stored at  for long term or  for short term (a few days).
-80 °C
4 °C
Flash freezing the virus particles in liquid nitrogen may increase the retention of their potency.
?. [Transduction with virus]
Seed iPSC cells so that they will reach 50 % confluency the next day.
?. [Transduction with virus]
Add virus to cells. The amount to add depends on how concentrated the virus is (adding ¼ or ½ of the total produced virus to cells is generally sufficient, see below for typical infection amounts).
?. [Transduction with virus]
Check next day for fluorescence by microscopy and the next time they are passaged by flow cytometry to check transduction efficiency.
?. [Transduction with virus]
For sgRNA constructs including puromycin resistance, add 0.8 ug/ml puromycin to select for cells with the sgRNA until they are at least 80 % confluent (typically within 2 passages).
?. [Day 1: Transfection]
Vortex gently before using.
?. [Day 3: Harvest]
Mix well by pipetting up and down 10x.
?. [Transduction with virus]
Check next day for fluorescence by microscopy, and the next time they are passaged by flow cytometry, to check transduction efficiency.
?. [Transduction with virus]
For sgRNA constructs including puromycin resistance, add 0.8 ug/ml puromycin to select for cells with the sgRNA until they are at least 80 % confluent (typically within 2 passages).