Oct 23, 2019
Lentiviral transduction of iPSCs with sgRNAs and sgRNA libraries
- Ruilin Tian1,
- Jason Hong1,
- Sydney Sattler1,
- Martin Kampmann1
- 1University of California, San Francisco
- Neurodegeneration Method Development Community
- KampmannLab
Protocol Citation: Ruilin Tian, Jason Hong, Sydney Sattler, Martin Kampmann 2019. Lentiviral transduction of iPSCs with sgRNAs and sgRNA libraries. protocols.io https://dx.doi.org/10.17504/protocols.io.8dfhs3n
Manuscript citation:
Tian et al (2019). CRISPR Interference-Based Platform for Multimodal Genetic Screens in Human iPSC-Derived Neurons. Neuron pii: S0896-6273(19)30640-3. [Epub ahead of print] PubMed PMID: 31422865.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 17, 2019
Last Modified: October 23, 2019
Protocol Integer ID: 28807
Keywords: Lentiviral transduction, Lentivirus, iPSC, sgRNA
Attachments
Materials
MATERIALS
DMEM, high glucoseThermo Fisher ScientificCatalog #11965092
Opti-MEM™ I Reduced Serum MediumThermo Fisher ScientificCatalog #31985070
TransIT®-Lenti Transfection ReagentMirus BioCatalog #MIR 6600
Lentivirus Precipitation SolutionALSTEM Cell AdvancementsCatalog #VC125
Equipment
12 ml Luer Lock Syringe
NAME
Syringe
TYPE
NORM-JECT ®
BRAND
4100.X00V0
SKU
LINK
Equipment
Filter, 0.45 µm
NAME
Sterile Syringe Filter
TYPE
Millex
BRAND
SLHV033RB
SKU
LINK
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Day 0: Seeding
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.
Incubate overnight.
Day 1: Transfection
Day 1: Transfection
Change 293T media with fresh DMEM.
Warm TransIT-Lenti Reagent to Room temperature .
Vortex gently before using.
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.
Note
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.
| Culture vessel | 48-well plate | 24-well plate | 12-well plate | 6-well plate | 10-cm dish | T75 flask | 15-cm dish | |
| Surface area | 1.0 cm^2 | 1.9cm^2 | 3.8 cm^2 | 9.6 cm^2 | 59 cm^2 | 75 cm^2 | 145cm^2 | |
| Complete growth medium | 263 μl | 0.5 ml | 1.0 ml | 2.0 ml | 10 ml | 15 ml | 30 ml | |
| Opti-Mem serum-free medium | 26 μl | 50 μl | 100 μl | 200 μl | 1.0 ml | 1.5 ml | 3.0 ml | |
| Transfer DNA (1 μg/μl stock) | 0.13 μl | 0.25 μl | 0.5 μl | 1.0 μl | 5 μl | 7.5 μl | 15 μl | |
| Packaging DNA Premix (1 μg/μl stock) | 0.13 μl | 0.25 μl | 0.5 μl | 1.0 μl | 5 μl | 7.5 μl | 15 μl | |
| TransIT-Lenti Reagent | 0.78 μl | 1.5 μl | 3 μl | 6 μl | 30 μl | 45 μl | 90 μl |
Add Opti-MEM into a sterile tube.
In another tube, mix Packaging DNA Premix and DNA.
Add the DNA mix to the Opti-MEM and mix gently.
Add TransIT-Lenti Reagent to the mixture and mix gently.
Incubate for 00:10:00 for transfection complexes to form.
Add all of the TransIT-Lenti:DNA complex mixture to the 293Ts dropwise and gently swirl to mix.
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
Day 3: Harvest
With a 12 ml syringe, take up the media/supernatant of the cells.
Put a 0.45 μm filter on the syringe and filter the supernatant into a fresh 15 ml conical tube.
Note
Change the filter if it becomes hard to push. Do not push too hard that bubbles are coming out.
Add 1:4 ratio of cold viral precipitation solution (e.g. 0.25 mL viral precipitation solution for 1 mL of viral supernatant).
Mix well by pipetting up and down 10x.
Incubate the viral supernatant at 4 °C for at least 04:00:00 and up to 3 days but no more than 3 days.
Cool down the centrifuge to 4 °C .
Spin down viral supernatant for 00:30:00 at 1500 x g .
The pellet will contain the virus.
Resuspend the pellet with 1 mL of your media of choice.
Virus can be aliquoted and stored at -80 °C for long term or 4 °C for short term (a few days).
Note
Flash freezing the virus particles in liquid nitrogen may increase the retention of their potency.
Transduction with virus
Transduction with virus
Seed iPSC cells so that they will reach 50 % confluency the next day.
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).
sgRNA library (15 cm dish)2 steps
A library prepared from a 15 cm dish typically infects 10 million iPSCs in one matrigel-coated T175 flask using 50 % of the produced virus.
Check next day for fluorescence by microscopy and the next time they are passaged by flow cytometry to check transduction efficiency.
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).