iNDI PiggyBac-TO-hNGN2 transfection protocol Version 1

Observe KOLF2.1 iPSCs under a phase contrast microscope to assess confluency and presence of cells debris. Dish should be dissociated at ~70% to 90% confluency.

Coat a well of 6 well plate to be used for transfection with 1mL of Matrigel solution, tilting to ensure coverage of entire surface area. Place in 37°C incubator for 0h 30m 0s .

Prepare E8 medium supplemented with CEPT and place in at Room temperature to warm during dissociation.

Aspirate culture medium from well/plate that should be dissociated and wash with PBS 1X.

Aspirate PBS and add half of culture volume of Accutase.

Transfer to 37°C incubator for 0h 10m 0s .

Meanwhile aspirate Matrigel from well and add 2mL culture medium E8 supplemented with CEPT.

When Incubation is ready, tilt the plate and pipet the accutase solution two to three times up and down the culture surface to break the colonies.

Quench the Accutase adding half of the culture volume of PBS.

Transfer to a new conical tube and rinse with more PBS the culture surface, combine with the cell solution in the tube.

Centrifuge 0h 5m 0s at 200 - 300 x g at Room temperature .

Aspirate supernatant and resuspend the cell pellet in culture medium E8 supplemented with CEPT.

Count cells and plate 1 - 1.5 x 106 cells ⁶ cells into a Matrigel-coated well. Gently swirl plate to evenly distribute cells.

Return plate to 37°C incubator.

After ~ 1h 0m 0s to 2h 0m 0s of plating cells , warm OptiMEM at Room temperature .

Prepare one tube with:

• 200µL of OptiMEM

• Total of 3µg of DNA mix in a 1:3 (Transposase:DNA) ratio

  Note

  EF1a-transposase sequence can be found in the materials section. Alternatively investigators can try the commercial superpiggybac transposase from SBI, but activity may be lower in iPSCs due to the use of a CMV promoter (which is rapidly silenced) rather than an EF1a promoter

• 5µL to 7.5µL of Lipofectamine Stem

  Note

  Vortex after adding every reagent to Opti-MEM

Incubate transfection mix for 0h 15m 0s to 0h 30m 0s at Room temperature .

Add transfection mix from step 15 to cells drop wise and immediately swirl the plate to evenly distribute the mix to cells.

Return plate to 37°C incubator.

Next day, check for nuclear BFP positive signal (for PB-TO-hNGN2-BFP plasmid). If a positive transfection occurred and the well is confluent, re-plate the cells and puromycin select 24-48 h after transfection (it all depends on the health and transfection efficiency of the cells) with 8µg/mL for 2 to 14 days.

Change media accordingly and expand cells when they reach 70-90% confluency.