TITLE: LIPID-MEDIATED TRANSFECTION OF iPSCs (Basic Protocol 2)
AUTHORS: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the lipid-mediated transfection of iPSCs maintained in E8 medium on Matrigel. While several lipid-based transfection reagents are commercially available, Lipofectamine Stem is used here because it is specifically optimized for delivery of DNA plasmids into hiPSCs. If preferred, similar results may also be achieved by electroporation or nucleofection, and other lipid reagents are available for </span><span style = "font-style:italic;">in vitro</span><span>-transcribed RNA or </span><span style = "font-style:italic;">in vitro-</span><span>translated ribonucleoproteins (RNPs). Furthermore, while this protocol provides the steps for a general transfection, specific details are provided below regarding insertion of the transgene cassettes relevant for neural differentiation (See </span><a href="https://www.protocols.io/view/basic-protocol-5-differentiation-of-i3neurons-5h6g39e" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Basic Protocols 5</span></a><span> and </span><a href="https://www.protocols.io/view/basic-protocol-7-differentiation-of-i3lmns-5tng6me" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">7</span></a><span>).</span></div></div>

[STEPS]
?. Grow a sufficient number of iPSCs for transfection and prepare cells as for an Accutase split (see Basic Protocol 1).
One or two wells of a 6-well dish at 80 % confluency should provide more than enough cells for one transfection.
?. Count the cells (Phelan & May, 2015), transfer 1.5 × 106 cells to a 15-ml conical tube, and centrifuge  at , .
0 Room temperature
?. Aspirate the supernatant and resuspend in  of E8 medium supplemented with  Y-27632 ROCK inhibitor.
2 ml
If iPSCs are normally maintained in a Flex medium, it is best to transition to regular E8 medium on the day of transfection to improve efficiency.
?. Pipet the medium and cells into 1 well of a 6-well dish pre-coated with Matrigel and return plate to the incubator. Gently shake the plate front-to-back and side-to-side.
?. Allow the cells to adhere in the incubator for  to  before adding the transfectionsolution.
?. For each transfection, add  of Opti-MEM and  of total DNA to one 1.5-ml microcentrifuge tube and vortex for  to . In a second tube, combine  of Opti-MEM and  of Lipofectamine Stem reagent, and vortex for  to .
100 µl
3 µg
100 µl
10 µl
For TALEN-mediated insertion to the AAVS1 or CLYBL locus, such as for the hNGN2 (Addgene, cat. no. 105840) and hNIL (Addgene. cat. no. 105841) differentiation cassettes, use a 2:1:1 ratio of   donor construct with   of each of the site-specific TALENs. For AAVS1:   of pTALdNC-AAVS1_T2 (Addgene, cat. no. 80496) and   of pTALdNC-AAVS1_T1 (Addgene, cat. no. 80495) per transfection. For CLYBL:   of pZT-C13-R1 (Addgene, cat. no. 62197) and   of pZT-C13-L1 (Addgene, cat. no. 62196) per transfection.
?. Combine the contents of the two tubes and vortex again for  to . Incubate thismixture for  at .
0 Room temperature
?. Retrieve the cells plated in steps 3 to 4, and, using a P200 pipet tip and repeat pipettor, add  of the complete transfection solution from step 6 dropwise, evenly across the surface of the well. Return the cells to the incubator overnight.
200 µl
?. after transfection, aspirate transfection medium and replace with fresh E8. If applicable, evaluate transfection efficiency by fluorescence microscopy.
All cells transfected with the hNGN2 (Addgene, cat. no. 105840 or 110492) and hNIL constructs (Addgene, cat. no. 105841 or 105842) will transiently express mCherry for 3 to 4 days, while only those cells with transgene insertion will maintain stable expression of mCherry for longer periods of time. See Basic Protocols 3 and 4 for options for enrichment and clonal isolation.
?. Change medium daily with normal culture medium, and wash with PBS if necessary to remove debris. Once the cells have reached 80 % confluency, they may be passaged to a new dish for expansion or used for enrichment or clonal selection.