Creating iPSC lines with Ribonucleoprotein (RNP): Nucleofection, Single-cell Sorting, Genotyping, and Line Maintenance Protocol

Cell Culture

Grow iPSCs in 1 well of a 6 well-plate until ~70-80% confluency.

Coating plates

Coat 1 x 6 well-plate with Matrigel.

• Matrigel is diluted in KO DMEM to make a working concentration of 80μl/ml (keep the Matrigel ice-cold)
• Matrigel coating volumes

1. 6 well plate: 1 mL
2. 12 well plate: 0.5 mL
3. 24 well plate: 0.25 mL
4. 48 well plate 0.125 mL
5. 96 well plate: 0.100 mL

Incubate the plate at 37°C for 0h 30m 0s.

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 ( 250µL ) in a 16 well nucleofector strip.

A	B
Nucleofector™ Supplement	45.45 µL
Nucleofector™ Solution	204.54 µL

Place On ice

Reconstitute 1.5nanomolar (nM) sgRNA in a BSC.

Add 15µL TE buffer to make 100micromolar (µM) sgRNA.

Store -20°C for long term storage.

Prepare RNP

For each nucleofection, mix a ratio of 1:3 (spCas9:guide)

sgRNA ( ) = , spCas9 ( ) = 100micromolar (µM)) = 1.2µL, spCas9 (40micromolar (µM)) = 1µL

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 0.5mL Accutase. Incubate the plate for 10-15 mins at 37°C. Quench the Accutase with 2.5mL of PBS.
• Transfer media into a 15 mL conical tube.
• Spin down the cells in a 15 mL conical for 0h 3m 0s at 800rpm,0h 0m 0s.
• Remove supernatant.
• Resuspend the cells in 1-2 mL of warm media.
  Note

  We use Accutase to obtain single cells.

Count cells.

Transfer ~350k cells in an Eppendorf tube (per reaction).

Prepare reaction.

Centrifuge the cells at 800rpm,0h 0m 0s for 0h 3m 0s. Aspirate supernatant.

Resuspend pellet in 20µL of P3 buffer.

Add 2µL of RNP (Cas9+sgRNA) to the Eppendorf tube.

If using two different RNPs, only add 1µL 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 2mL of media.

Recover Cells

Add 80µL 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 50µL/well of the cells to 2 wells of a 6 well-plate.

Gently rock the plate and incubate 0h 3m 0s at 37°C.

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).

At 70-80% confluency, collect the pooled population of cells.

Incubate cells in 0.5mL Accutase. Incubate the plate for ~5-10 mins at 37°C.

Quench the Accutase with 2.5mL of PBS.

Transport 1.5mL/conical tube of the cells to 2 x 15 mL conical tube.

Spin down the cells at 800rpm,0h 0m 0s for 0h 3m 0s.

Aspirate the supernatant.

Freezing the pool.

Resuspend 1 of the pellets in 1mL 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 24h 0m 0s.

• After 24-48 hours, move the vial to liquid nitrogen.

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.

Preparing a 96 well-plate

Coat a 96 well-plate with 100µL/well of Matrigel.

Allow the plate to incubate wi for at least 0h 30m 0s at 37°C .

Prepare 20mL 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

Aspirate the Matrigel from the 96 well-plate and add 100µL/well of media to each well.

Set the plate back into the incubator.

Preparing Cells

Wash the well with PBS.

Dilute cells to a 1.5 million cells/500µL.

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.

Add 0.5mL Accutase.

Incubate the plate for ~10-15 mins at 37°C.

Quench the Accutase with 2.5mL of PBS.

Transport cells into a 15 mL conical tube.

Spin down the cells for 0h 3m 0s at 800rpm,0h 0m 0s.

Remove supernatant.

Resuspend the cells in1-2mLof warm media.

Count cells.

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.

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.

Day 4

Prepare fresh media without ROCK Inhibitor. For 12mL

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 100µL/ well of the new media into each well of a 96 well-plate.

Check there is 1 colony/well. Ensure colonies are growing.

Day 6

Prepare fresh media without ROCK Inhibitor and Clone R. For 12mL.

A	B
mTeSR Plus	12 mL
Anti-Anti (100x)	120 µL

Aspirate spent media using a multichannel aspirator.

Pipette 100µL/ well of the new media into each well of a 96 well-plate.

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 100µL/ 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.

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

If there are less than 24 clones, then you will passage the cells into 2 x 24- well-plates.

Prepare the 24 or 48 well-plates.

Matrigel coat the 24 or 48 well-plates.

Incubate for at least 0h 30m 0s at 37°C.

Prepare media. For 25mL

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 250µL/well
• For 24 well-plates: add 500µL/well

Clump passage the clones (row-by-row)

Aspirate spent media from one row of the 96 well-plates.

Continue row-by-row until the entire plate is passaged.

Pipette 50µL/well of ReLeSR.

Incubate at Room temperature for 0h 0m 45s.

Aspirate ReLeSR from cells.

Incubate the 96 well-plate at 37°C for 0h 3m 0s.

Pipette 100µL/well of mTeSR Plus w/ ROCK inhibitor to each well in the row.

Resuspend cells by pipetting up and down.

Move 35µL into one well of the new plates and 65µL into another well of the other new plate.

Once a row is passaged, disperse the cells by rocking in all four directions.

Day 1 post passaging

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 200µL/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 50µL/well of Quick Extract.

ii. Scrape the bottom of the well to detach cells.

iii. Mix by vortexing for 0h 0m 15s.

iv. Transfer cells in Quick Extract to labeled PCR tubes.

v. Incubate the samples at 65°C for 0h 6m 0s using a Thermocycler.

vi. Mix by vortexing.

vii. Incubate the samples at 98°C for 0h 2m 0s using a Thermocycler.

(Optional) Freezing the clones.

Add Mineral Oil

• For 96 well-plates: 100µL/well
• For 48 well-plates: 200µL/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.

Obtain a Styrofoam box and clean it with ethanol.

Grow the plate until clones are 70-80% confluent.

Aspirate spent media.

Wash cells with 200µL/well of PBS. Aspirate

Add 100µL/well of ReLeSR.

Incubate at Room temperature for 0h 0m 45s.

Aspirate and incubate plate at 37°C for 0h 3m 0s.

Add CryoStor

• For 96 well-plates: 100µL/well.
• For 48 well-plates: 200µL/well.

(Optional) Thawing clones from frozen 96/48 well-plate.

Coat 12 well plates with 0.5mL/well of Matrigel.

• Incubate the plate at 37°C for at least 0h 30m 0s.

Resuspend the pellet in 1mL of media.

Pipette the cells into 1 well of a 12 well-plate.

Place plate in incubator.

Label Eppendorf tubes with the clone number of the clones that you will move forward with

Prepare Media. For 12mL

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 0h 10m 0s or until edges of the plate are thawed.

Pipette warm PBS onto wells that you want to thaw.

Pipette the cells into their respective Eppendorf tube.

Centrifuge at 800rpm,0h 0m 0s for 0h 3m 0s.

Aspirate the supernatant

Obtaining gene sequences: National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/)

Storing and aligning sequences: SnapGene

Designing primers: Primer Blast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/)

Ordering primers: Integrated DNA Technologies (www.idtdna.com/)

• Products and Services → Custom DNA Oligos → DNA Oligos (order now)

Check for off targets: Basic Local Alignment Search Tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi)

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.

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.