May 20, 2022
Creating iPSC lines with Ribonucleoprotein (RNP): Nucleofection, Single-cell Sorting, Genotyping, and Line Maintenance Protocol
- Kamaljot Gill1,2,
- Aradhana Sachdev2,
- Bruce Conklin2,
- Claire D Clelland1,2,3
- 1University of California, San Francisco, Weill Institute for Neurosciences, USA;
- 2Gladstone Institutes, San Francisco, CA, USA;
- 3Department of Neurology, University of California, San Francisco, San Francisco, CA, USA
Protocol Citation: Kamaljot Gill, Aradhana Sachdev, Bruce Conklin, Claire D Clelland 2022. Creating iPSC lines with Ribonucleoprotein (RNP): Nucleofection, Single-cell Sorting, Genotyping, and Line Maintenance Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2oeopv1y/v1
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: May 10, 2022
Last Modified: May 20, 2022
Protocol Integer ID: 62319
Keywords: iPSC lines , Ribonucleoprotein (RNP), gene editing , nucleofection, single-cell sorting, FACS, genotyping, spCas9, Line Maintenance, Stem Cells, excision
Abstract
This protocol describes how to perform gene editing on human induced pluripotent stem cells (iPSCs) via ribonucleoprotein (RNP) and how to the isolate lines with the desired excision. It describes nucleofection, single cell sorting via FACS, genotyping, and the maintenance of the cell lines throughout the process. This protocol is optimized for spCas9.
Attachments
Guidelines
Schematic describing the flow of steps in creating iPSC cell lines with RNP
Materials
Reagents:
P3 Primary Cell 96-well Nucleofector™ Kit (960 RCT)LonzaCatalog #V4SP-3096
ReLeSR™ 100 mL
Stemcell TechnologiesCatalog #5872
PBS, pH 7.4Thermo FisherCatalog #10010023
ACCUTASE™ 100 mL
Stemcell TechnologiesCatalog #7920
mTeSR™ PlusStemcell TechnologiesCatalog #100-0276
CloneR™ 10 mL
Stemcell TechnologiesCatalog #5888
KnockOut™ DMEMThermo Fisher ScientificCatalog #10829018
Growth Factor Reduced (GFR) Matrigel® phenol red-freeCorningCatalog #356231
CryoStor® CS10Stemcell TechnologiesCatalog #07930
QuickExtract DNA Extraction SolutionLucigenCatalog #QE09050
Antibiotic-Antimycotic (100X)Thermo FisherCatalog #15240062
Y-27632SelleckchemCatalog #S1049
Olympus Plastics 28-154 35µm Strainer CapsGenesee ScientificCatalog #28-155
Sigma IsopropanolCatalog #190764-4L
Mineral OilSigma AldrichCatalog #M8410-100ML
| A | B | C | |
| Reagent | Company | Catalog # | |
| P3 Primary Cell 96-well Nucleofector™ Kit (96 RCT) | Lonza | V4SP-3096 | |
| ReLeSR™ | Stemcell | 5872 | |
| PBS, pH 7.4 | ThermoFisher | 10010023 | |
| Accutase™ | Stemcell | 7920 | |
| mTeSR™ Plus | Stemcell | 100-0276 | |
| CloneR™ | Stemcell | 5888 | |
| KnockOut™ DMEM | ThermoFisher | 10829018 | |
| Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-free, LDEV-free, 10 mL | Corning | 356231 | |
| CryoStor® CS10 | Stemcell | 7930 | |
| QuickExtract™ DNA Extraction Solution | Lucigen | QE09050 | |
| Antibiotic-Antimycotic (100X) | ThermoFisher | 15240062 | |
| ROCK1 Inhibitor (Y-27632 2HCl) | Selleckchem | S1049 | |
| Olympus Plastics 28-154, 35µm Strainer Caps | Genesee | 28-155 | |
| 2-Propanol | Sigma | 190764-4L | |
| Mineral Oil | Sigma | M8410-100ML | |
| Cas9-NLS Purified Protein | MacroLabs |
Equipment:
Equipment
4D-Nucleofector Core Unit
NAME
Control system for performing nucleofection
TYPE
Lonza
BRAND
AAF-1002B
SKU
LINK
Equipment
4D-Nucleofector X Unit
NAME
Supports Nucleofection® of various cell numbers in 100 µL cuvettes or 20 µL 16-well strips
TYPE
Lonza
BRAND
AAF-1002X
SKU
https://bioscience.lonza.com/lonza_bs/CH/en/Transfection/p/000000000000276883/4D-Nucleofector-X-Unit
LINK
Equipment
Mr. Frosty Freezing Container
NAME
freezing container
TYPE
ThermoFisher Scientific
BRAND
5100-0001
SKU
LINK
Equipment
Costar® vacuum aspirator
NAME
MilliporeSigma
BRAND
CLS4931
SKU
LINK
| A | B | C | |
| Equipment | Company | Catalog # | |
| BD FACS Aria Fusion | Beckton Dickinson | - |
Nucleofecting
Nucleofecting
Cell Culture
Grow iPSCs in 1 well of a 6 well-plate until ~70-80% confluency.
Note
At this confluency in a 6 well-plate, there should be at least 1.5 million cells.
Coating plates
Coat 1 x 6 well-plate with Matrigel.
- Matrigel is diluted in KO DMEM to make a working concentration of 80 μl/ml (keep the Matrigel ice-cold)
- Matrigel coating volumes
- 6 well plate: 1 mL
- 12 well plate: 0.5 mL
- 24 well plate: 0.25 mL
- 48 well plate 0.125 mL
- 96 well plate: 0.100 mL
Incubate the plate at 37 °C for 00:30:00 .
30m
Turn on the Lonza 4D-Nucleofector.
Prepare Media
For 20 mL:
| A | B | |
| mTeSR Plus | 18 mL | |
| Clone R (10X) | 2 mL | |
| 10mM ROCK Inhibitor (RI) | 20 µL |
Prepare P3 Buffer.
For 12 reactions ( 250 µL ) in a 16 well nucleofector strip.
| A | B | |
| Nucleofector™ Supplement | 45.45 µL | |
| Nucleofector™ Solution | 204.54 µL |
Place On ice
Note
Once made, you may store P3 buffer at 4 °C for up to 3 months.
Reconstitute 1.5 nanomolar (nM) sgRNA in a BSC.
Add 15 µL TE buffer to make 100 micromolar (µM) sgRNA.
Store -20 °C for long term storage.
Prepare RNP
For each nucleofection, mix a ratio of 1:3 (spCas9:guide)
sgRNA (100 micromolar (µM) ) = 1.2 µL , spCas9 (40 micromolar (µM) ) = 1 µL
Incubate RNPs at Room temperature for 10-15 mins.
Prepare cells (below are the instructions for 1 well of a 6-well plate)
Wash the well with PBS.
Detach cells from plate with Accutase.
- Incubate cells in 0.5 mL Accutase. Incubate the plate for 10-15 mins at 37 °C . Quench the Accutase with 2.5 mL of PBS.
- Transfer media into a 15 mL conical tube.
- Spin down the cells in a 15 mL conical for 00:03:00 at 800 rpm .
- Remove supernatant.
- Resuspend the cells in 1-2 mL of warm media.
Note
We use Accutase to obtain single cells.
3m
Count cells.
Transfer ~350k cells in an Eppendorf tube (per reaction).
Prepare reaction.
Centrifuge the cells at 800 rpm for 00:03:00 . Aspirate supernatant.
3m
Resuspend pellet in 20 µL of P3 buffer.
Add 2 µL of RNP (Cas9+sgRNA) to the Eppendorf tube.
If using two different RNPs, only add 1 µL of each because only half of each RNP is needed.
Transfer each sample to one well of a 16 strip nucleofector cuvette/strip. Check lid to make sure it is in the correct orientation.
Tap nucleofector strip on surface to distribute sample and pop any bubbles.
- If needed, use a P20 pipette tip to gently pop any bubbles.
Nucleofecting
Set up the reaction on 4D-Nucleofector Core X - Unit
- Select wells being used in your script
- Select P3 buffer setting
- Select 16-well strip
- Select Pulse Code = DS138
Place the nucleofector strip in the nucleofector and hit start.
Green plus sign should appear if reaction is successful.
Place the nucleofector strip in the hood (do not spray it with EtOH) and incubate at Room temperature for 5-10 mins.
Prepare plate
Obtain the 6 well-plate that was coated with Matrigel earlier.
Aspirate Matrigel.
Replace with 2 mL of media.
Recover Cells
Add 80 µL of media to each well of the nucleofector strip to recover the cells.
Slowly pipette up and down to ensure cells are properly dispersed.
Transfer cells to plate.
For each nucleofection, transfer 50 µL /well of the cells to 2 wells of a 6 well-plate.
Gently rock the plate and incubate Overnight at 37 °C .
3m
Maintain cells.
Grow the cells until they are 70-80% confluency (approximately 3-4 days).
When cells are at 70-80% confluency, you may
- Harvest one well for sorting.
- Harvest one well for freezing and genotyping (lift the well and pellet into 2 separate conical tubes).
Harvesting Cells for Freezing and Genotyping:
Harvesting Cells for Freezing and Genotyping:
At 70-80% confluency, collect the pooled population of cells.
Incubate cells in 0.5 mL Accutase. Incubate the plate for ~5-10 mins at 37 °C .
Quench the Accutase with 2.5 mL of PBS.
Transport 1.5 mL /conical tube of the cells to 2 x 15 mL conical tube.
Spin down the cells at 800 rpm for 00:03:00 .
3m
Aspirate the supernatant.
Freezing the pool.
Resuspend 1 of the pellets in 1 mL of ice-cold CryoStor.
Transfer cells into a cryovial.
Transport vial to a Mr. Frosty with 2-propanol.
Place Mr. Frosty at -80 °C to freeze for 24:00:00 .
- After 24-48 hours, move the vial to liquid nitrogen.
1d
Genotyping pool (go to the Genotyping section for more information)
Store the remaining pellet in the conical tube at -20 °C until ready to extract DNA.
Perform an excision PCR with an unedited line as a negative control.
Note
This is an important check that can save you time later on. Genotyping the pool informs you that you have any excision in any cells.
Single Cell Sorting by FACS
Single Cell Sorting by FACS
Preparing a 96 well-plate
Coat a 96 well-plate with 100 µL /well of Matrigel.
Allow the plate to incubate wi for at least 00:30:00 at 37 °C .
30m
Prepare 20 mL of Media (mTeSR Plus + ROCK Inhibitor (1,000X) + Clone R (10X)+ Anti-Anti (100X)).
| A | B | |
| mTeSR Plus | 18 mL | |
| CloneR (10X) | 2 mL | |
| Anti-Anti (100X) | 200 µL | |
| 10mM ROCK Inhibitor | 20 µL |
Note
If using alternative media, it must NOT be more than 2% FBS
Aspirate the Matrigel from the 96 well-plate and add 100 µL /well of media to each well.
Set the plate back into the incubator.
Preparing Cells
Wash the well with PBS.
Add 0.5 mL Accutase.
Incubate the plate for ~10-15 mins at 37 °C .
Quench the Accutase with 2.5 mL of PBS.
Transport cells into a 15 mL conical tube.
Spin down the cells for 00:03:00 at 800 rpm .
3m
Remove supernatant.
Resuspend the cells in1-2 mL of warm media.
Count cells.
Dilute cells to a 1.5 million cells/500 µL .
Note
For single - sorting, 1.5 million cells in 500 µL of media is an appropriate concentration.
Pass cells through a filter mesh (strainer cap) using a P1000.
- Place a filter mesh on top of a FACS collection tube.
- Replace filter mesh with cap for collection tube.
Note
You can directly press the tip against the mesh and pipette the cell solution into the collection tube.
Transport cells to FACS machine.
Seal the 96 well-plates with media with parafilm.
Seal the collection tube with parafilm.
Clean a large container with ethanol.
Place the 96 well-plate and collection tube into the container.
FAC Sorting
Perform single cell sorting with aBD FACS Aria Fusion (Beckton Dickinson), equipped with 355, 405, 488, 561 and 640 nm lasers.
Note
The QC alignment of each laser should be verified with Cytometer Setup and Tracking Beads (Becton Dickinson) before sample acquisition.
Set forward a scatter threshold of 15,000 to eliminate debris from list mode data, and fix the number of events to be collected.
In certain experiments mCherry fluorescence (excitation 561 nm, emission 610 nm) can be used to define sorting parameters.
Drop delay determination and 96 well plate set-up setup using Accudrop beads (Becton Dickinson).
Use forward scatter area versus height and side scatter area versus height gates to make the single cell determination. The specifications of the sort layout include single cell precision, 96 well collection device and target event of 1.
Quarantining Cells (Day 0)
Move cells to the quarantine incubator (or a separate incubator from other cell lines) during the duration of 7-day Anti-Anti treatment and before confirming the cells are mycoplasma negative
- Perform a mycoplasma test between Day 3 and Day 7.
Days 1-3
Do not change media.
Ensure there is 1 cell/well and there is no contamination.
Note
Note 1: Cells will be hard to see for the first few days.
Note 2: Expect a ~40% cell survival for single cells.
Day 4
Prepare fresh media without ROCK Inhibitor. For 12 mL
| A | B | |
| mTeSR Plus | 10.8 mL | |
| Clone R (10X) | 1.2 mL | |
| Anti-Anti (100X) | 120 µL |
Aspirate spent media using a multichannel aspirator.
Pipette 100 µL / well of the new media into each well of a 96 well-plate.
Note
Be careful not to cross contaminate. These are individual clones.
Check there is 1 colony/well. Ensure colonies are growing.
Day 6
Prepare fresh media without ROCK Inhibitor and Clone R. For 12 mL .
| A | B | |
| mTeSR Plus | 12 mL | |
| Anti-Anti (100x) | 120 µL |
Aspirate spent media using a multichannel aspirator.
Pipette 100 µL / well of the new media into each well of a 96 well-plate.
Note
Be careful not to cross contaminate. These are individual clones.
Check there is 1 colony/well. Ensure colonies are growing.
Perform a mycoplasma test during this stage or earlier.
Day 8 and later
If the cells are 70-80% confluent or begin to grow on top of each other, move onto the next step. If not, continue with this step.
If the cells are not ready, continuing feeding but with just mTeSR Plus
- Aspirate spent media using a multichannel aspirator.
- Pipette 100 µL / well of mTeSR Plus into each well of a 96 well-plate.
Note
Be careful not to cross contaminate. These are individual clones.
- Check there is 1 colony/well. Ensure colonies are growing.
Keep cells in 96 well-plate for 1-7 more days or until the clones can be passaged to a smaller plate format.
Passaging and Maintaining the Clones
Passaging and Maintaining the Clones
Identify the surviving clones.
Using a microscope and marker, count and label the wells of the 96 well-plate where there are surviving clones.
If there are between 24 and 48 clones, then you will passage the cells in 2 x 48 well-plates
Note
One plate is to keep the clone in culture and the other plate is for genotyping
If there are less than 24 clones, then you will passage the cells into 2 x 24- well-plates.
Note
One plate is to keep the clone in culture and the other plate is for genotyping
Prepare the 24 or 48 well-plates.
Matrigel coat the 24 or 48 well-plates.
Incubate for at least 00:30:00 at 37 °C .
30m
Prepare media. For 25 mL
| A | B | |
| MteSR Plus | 25 mL | |
| 10mM RI µL | 25 µL |
Aspirate the Matrigel from plates.
Add media to plates
- For 48 well-plates: add 250 µL /well
- For 24 well-plates: add 500 µL /well
Clump passage the clones (row-by-row)
Aspirate spent media from one row of the 96 well-plates.
Pipette 50 µL /well of ReLeSR.
Incubate at Room temperature for 00:00:45 .
45s
Aspirate ReLeSR from cells.
Incubate the 96 well-plate at 37 °C for 00:03:00 .
3m
Pipette 100 µL /well of mTeSR Plus w/ ROCK inhibitor to each well in the row.
Resuspend cells by pipetting up and down.
Move 35 µL into one well of the new plates and 65 µL into another well of the other new plate.
Note
Note 1: Be sure that you pipette into the same pattern to ensure the 2 plates are identical.
Note 2: It is possible to add more cells to one plate than the other to harvest the more confluent plate first.
Once a row is passaged, disperse the cells by rocking in all four directions.
Continue row-by-row until the entire plate is passaged.
Day 1 post passaging
Note
Most of the time, at least one plate will be 70-80% confluent the next day. This plate can be harvested for genotyping. The other plate may be a little less confluent and should can be maintained until it is 70-80% confluent (usually the day after).
Note
Genotyping (at least performing the excision PCR) can occur within the day and the clones with the preferred edit can be identified.
If one plate is at least 70-80% confluent, move to the next step. Otherwise, maintain the plates.
Harvesting one plate to genotype the clones.
Aspirate the spent media.
Wash the cells with 200 µL /well of PBS.
Aspirate the PBS.
Seal the sides of the plate with parafilm to avoid evaporation
Store the plate at -20 °C and harvest DNA later or extract DNA immediately using Quick Extract.
Quick Extract Protocol.
i. Add 50 µL /well of Quick Extract.
ii. Scrape the bottom of the well to detach cells.
Note
Time can be saved by performing the above steps to the entire row before moving to the steps below (one row at a time).
iii. Mix by vortexing for 00:00:15 .
iv. Transfer cells in Quick Extract to labeled PCR tubes.
Note
If the QE is very viscous at this step, use more QE to obtain a more fluid consistency.
v. Incubate the samples at 65 °C for 00:06:00 using a Thermocycler.
vi. Mix by vortexing.
vii. Incubate the samples at 98 °C for 00:02:00 using a Thermocycler.
8m 15s
(Optional) Freezing the clones.
Note
There is a probability that some clones will be lost via the freezing and thawing process.
Obtain a Styrofoam box and clean it with ethanol.
Grow the plate until clones are 70-80% confluent.
Aspirate spent media.
Wash cells with 200 µL /well of PBS. Aspirate
Add 100 µL /well of ReLeSR.
Incubate at Room temperature for 00:00:45 .
45s
Aspirate and incubate plate at 37 °C for 00:03:00 .
3m
Add CryoStor
- For 96 well-plates: 100 µL /well.
- For 48 well-plates: 200 µL /well.
Add Mineral Oil
- For 96 well-plates: 100 µL /well
- For 48 well-plates: 200 µL /well
Seal the plate with parafilm.
Place the plate in the Styrofoam box.
Carefully transfer the box to a -80 °C freezer.
The cells are stable for up to 1 month at -80 °C .
(Optional) Thawing clones from frozen 96/48 well-plate.
Coat 12 well plates with 0.5 mL /well of Matrigel.
- Incubate the plate at 37 °C for at least 00:30:00 .
30m
Label Eppendorf tubes with the clone number of the clones that you will move forward with
Prepare Media. For 12 mL
| A | B | |
| mTeSR Plus | 12 mL | |
| 10mM RI µL | 12 µL |
Warm PBS to 37 °C .
Place the frozen plate on paper towels and place it in the 37 °C incubator for 00:10:00 or until edges of the plate are thawed.
10m
Pipette warm PBS onto wells that you want to thaw.
Pipette the cells into their respective Eppendorf tube.
Centrifuge at 800 rpm for 00:03:00 .
3m
Aspirate the supernatant
Note
Pellet will be too small to visualize, but it is there!
Resuspend the pellet in 1 mL of media.
Pipette the cells into 1 well of a 12 well-plate.
Place plate in incubator.
Genotyping
Genotyping
Note
In order to save time and reagents, it is recommended to perform genotyping in various stages and reduce the total number of potential clones throughout the process. Specifically, perform the excision PCR on all the clones, then only perform 5’ and 3’ cut site PCR on clones that have the correct excision band. Likewise, only maintain clones which pass the various genotyping stages.
Note
You may design and manage the primers using a various set of resources, but the following tools are recommended.
Obtaining gene sequences: National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/)
Storing and aligning sequences: SnapGene
- Products and Services → Custom DNA Oligos → DNA Oligos (order now)
Perform an excision PCR (perform on all clones).
Designing excision primers
- These set of primers should bind outside of both the cut sites.
- Length: After the excision, the expected length of the band should be between 100 bp and 1000 bp.
Expectations
- Unedited line: A large band or no band because of the size of the excision.
- Homozygous excision: Expected band size.
- Heterozygous excision: Expected band size and the unedited band (unless there is no band).
Sanger Sequence
- Save some PCR product to sanger sequence at a later time.
Perform 5’ cut site excision PCR (perform only on clones that have the correct excision band).
Design 5’ cut site primers.
- One of the primers should bind on the 5’ outside of the cut site and the other primer should bind within the excision region.
- Length: If the excision did not occur, the expected length of the band should be between 100 bp and 1000 bp. With the excision, we expect no band
Expectations
- Unedited line: Expected band size
- Homozygous excision: No band
- Heterozygous excision: Expected band size
Perform 3’ cut site excision PCR (perform only on clones that have the correct excision band).
Design 3’ cut site primers.
- One of the primers should bind 3’ outside of the cut site and the other primer should bind within the excision region.
- Length: If the excision did not occur, the expected length of the band should be between 100 bp and 1000 bp. With the excision, we expect no band.
Expectations
- Unedited line: Expected band size.
- Homozygous excision: No band.
- Heterozygous excision: Expected band size.
Karyotyping and Freezing
Karyotyping and Freezing
Send one or two clones with correct excisions to karyotype.
Freeze a few vials (3-4) of all clones you are karyotyping.
Expand and freeze 10+ vials the clone with the correct excision and normal karyotype.