Table 3.

Troubleshooting Guide

Step	Problem	Solution
Basic Protocol, step 29	Increased cell‐death after transfection	Some degree of cell‐death is expected after the transfection of the cells using these electroporation parameters. However, there should be attached cells 24 hr post plating.
Basic Protocol, step 31 (annotation)	No hiPSC colonies appearing (with transfection efficiency >0.1%)	Ensure your UDCs are low passage and proliferative.
Basic Protocol, step 31 (annotation)	hiPSC colonies remain small due to fully confluent non‐reprogrammed UDCs	Carefully remove non‐reprogrammed UDCs surrounding the hiPSC colony by scraping with a pipette‐tip without damaging the colony. Change medium and keep in culture for a couple of days until hiPSC colony is ready for picking.
Basic Protocol, step 43	Transfection efficiency below 0.1%	Repeat the experiment ensuring that: TF medium does not contain antibiotics. Cells appeared as single cells before transfection Transfect the correct number of viable cells. The srRNA has not been degraded. Use RNase‐free tubes and tips. Thaw srRNA on ice shortly before use. Avoid repeated freezing and thawing. No error or spark was observed using the Neon transfection system
Support Protocol 1, step 15	Low fluorescent intensity for one or more antibodies	There might be batch‐to‐batch variation for pre‐labeled antibodies. Test different batches and determine the optimal dilution.
Support Protocol 2, step 7 & 8	Cells are detached from coverslips after this step	Avoid adding DPBS and 2% PFA at high speed directly onto the cells. As the cells differentiate and become dense, they tend to detach more easily (as a sheet). Add fluids gently against the well wall.