TITLE: Nucleofection of hPSCs
AUTHORS: Hanqin  Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the standard procedure for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) into human pluripotent stem cells (hPSCs) using nucleofection.


General notes
Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.

[STEPS]
1. For each nucleofection, prepare 20 µl nucleofection solution by mixing 16.4 µl solution I and 3.6 µl supplement. 
4. Re-suspend cell pellets in nucleofection solution. Mix using P1000 or P200 tips to ensure single cell suspension. 
5. Transfer 20 µl cell suspension to the microcentrifuge tube containing the materials to be nucleofected. Mix well.
6. Transfer the entire volume to one cuvette in the 16-cuvette strip. Mark the cuvette used on the lid.
7. Gently stamp the strip on bench to help the small amount of cell suspension enter the narrow space between the metal paddles properly.
8. Nucleofect with program, P3 primary cell, CA137
 

9. After nucleofection, immediately add 150 µl hPSCs medium or feeder-free medium + Rock Inhibitor to the cuvette according to the culturing system that will be used. 

For a detailed description of hPSCs medium, refer to the protocol "Preparing MEF-cultured hPSCs for nucleofection" in the collection "Nucleofection (Amaxa) and electoporation (Biorad) of hPSCs. A link to this collection can be found in the title section of this protocol, located above. 

For a detailed description of Feeder-free medium + Rock Inhibitor, refer to the collection "Feeder-free culturing of hPSCs;" dx.doi.org/10.17504/protocols.io.b4mcqu2w

10. Pipet thoroughly using a P200 tip
11. Transfer all cells to a new microcentrifuge tube
12. Transfer proper amount of cells to MEFs plate or VTN/Matrigel/Geltrex-coated plate

For a detailed protocol on plating hPSCs on MEFS refer to the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture;" dx.doi.org/10.17504/protocols.io.b4pbqvin

For a detailed protocol on coating plates with VTN/Matrigel/Geltrex, refer to the collection "Feeder-free culturing of hPSCs;" dx.doi.org/10.17504/protocols.io.b4mcqu2w

13. Place the plate in the low oxygen incubator, 3% O2, 5% CO2.
2. Prepare plasmids, RNA, or RNP as described in their associated protocols in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs." A link to this collection can be found in the title section of this protocol, located above. The specific protocol titles for each preparation type are listed below. 

Plasmids: Preparing plasmids for nucleofection
RNA: Preparing mRNA for nucleofection
RNP: In vitro assembling of RNP for nucleofection
3. Prepare cells for nucleofection as described in protocols  “Preparing MEF-cultured hPSCs for nucleofection” or “Preparing feeder-free hPSCs for nucleofection," which are both part of the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs." A link to this collection can be found in the title section of this protocol, located above