Oct 23, 2019
iPSC editing with TALENs
- Ruilin Tian1,
- Jason Hong1,
- Martin Kampmann1
- 1University of California, San Francisco
- Neurodegeneration Method Development Community
- KampmannLab
Protocol Citation: Ruilin Tian, Jason Hong, Martin Kampmann 2019. iPSC editing with TALENs. protocols.io https://dx.doi.org/10.17504/protocols.io.8dahs2e
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: 28802
Keywords: iPSC, TALENs, CRISPR interference, CRISPRi, CROP-seq, Perturb-Seq, essential genes, functional genomics, high-content microscopy, neuron, single-cell RNA sequencing, stem cell
Attachments
Materials
MATERIALS
pZT-C13-L1addgeneCatalog #62196
pZT-C13-R1addgeneCatalog #62197
DPBS (no Ca, no Mg)ThermofisherCatalog #14190144
Essential 8™ MediumGibco - Thermo Fisher ScientificCatalog #A1517001
StemPro™ Accutase™ Cell Dissociation ReagentThermo Fisher ScientificCatalog #A1110501
Lipofectamine™ Stem Transfection ReagentThermo Fisher ScientificCatalog #STEM00008
StemFlex™ MediumThermo Fisher ScientificCatalog #A3349401
MatrigelCorningCatalog #356231
KnockOut™ DMEMThermo Fisher ScientificCatalog #10829018
Opti-MEM™ I Reduced Serum MediumThermo Fisher ScientificCatalog #31985070
Y-27632 dihydrochloride (Rock Inhibitor)R&D SystemsCatalog #1254/10
- BCL-XL plasmid (pEF1-BCL-XL-wpre-polyA P1102): Gift from Xiaobing Zhang, described in PMID: 30239926
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Pre-coating 6 well plate
Pre-coating 6 well plate
Pre-coat 6 well plate with Matrigel (diluted 1:50 with Knockout DMEM) by adding 1 mL to each well.
Let it sit in incubator for at least 00:20:00 .
Passaging and Plating iPSC Cells
Passaging and Plating iPSC Cells
Grow iPSCs until they are ~ 85 % confluent in one well of a 6 well plate or larger format.
Remove old medium and wash iPSCs with DPBS 1x.
Add Accutase for 00:03:00 – 00:05:00 to lift the cells.
Singularize the cells by gently pipetting them up and down several times.
In a 15 ml conical tube, add DPBS and then add the lifted cells in Accutase.
Spin down at 200 x g for 00:05:00 .
Aspirate supernatant and resuspend cells in Stemflex.
Perform cell count.
Remove the Matrigel in the pre-coated plate and add appropriate amount of Stemflex medium with Rock inhibitor (1000x).
Re-seed 0.5 M cells into a 6 well plate so that it can reach ~ 60 % confluency the next day.
Put the plate in incubator and culture overnight.
Transfection
Transfection
The following day, change the media with new E8 media.
Prepare the following mixes:
Tube 1:
- 100 µL Opti-Mem
- 10 µL Lipofectamine Stem Reagent:
Tube 2:
- 100 µL Opti-Mem
- DNA mix (0.5 µg – 5.0 µg total)
Optimized TALENS ratio
- 1.5 µg Your Plasmid of choice
- 0.75 µg TALENS L
- 0.75 µg TALENS R
- 0.3 µg BCL-XL
Add tube 2 to tube 1 and mix well.
Incubate the mixture for 00:10:00 .
Add the whole mixture to your cells in the 6 well plate.
Incubate overnight and change the media to Stemflex the next day.
If confluent, passage and expand them into 10 cm plate.
When confluent in 10 cm plate, continue to selection protocol.