TITLE: 3.2 Nucleofection of iPSCs
AUTHORS: Evelyn J. Sauter, Lisa K. Kutsche, Simon D. Klapper, Volker Busskamp

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is part 3.2 of the "</span><a href="https://www.protocols.io/private/C65DB81838BC11EBA61A0A58A9FEAC2A/protocols" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Induced Neurons for the Study of Neurodegenerative and Neurodevelopmental Disorders</span></a><span>"  collection of protocols.</span></div><br/><div class = "text-block"><span style = "font-weight:bold;">Collection Abstract:</span><span> Patient-derived or genomically modified human induced pluripotent stem cells (iPSCs) offer the opportunity to study neurodevelopmental and neurodegenerative disorders. Overexpression of certain neurogenic transcription factors (TFs) in iPSCs can induce efficient differentiation into homogeneous populations of the disease-relevant neuronal cell types. Here we provide protocols for genomic manipulations of iPSCs by CRISPR/Cas9. We also introduce two methods, based on lentiviral delivery and the piggyBac transposon system, to stably integrate neurogenic TFs into human iPSCs. Furthermore, we describe the TF-mediated neuronal differentiation and maturation in combination with astrocyte cocultures.</span></div></div>

[STEPS]
?. [Nucleofection of iPSCs]
In order to electroporate piggyBac and transposase vectors into iPSCs in suspension, use the X-Unit of the 4D- Nucleofector™ System in combination with the P3 Primary Cell 4D-Nucleofector™ X Kit according to the manufacturer’s guidelines.
?. [Nucleofection of iPSCs]
First of all, prepare the DNA, the Nucleofector™ solution and the cell culture plates. For a nucleofection reaction in 100 μl cuvettes, mix  and  in less than  volume (maximum  of the final sample volume) in a 1.5 ml tube.
[piggyBac vector]
[transposase vector]
10 µl
?. [Nucleofection of iPSCs]
Switch on the X-Unit of the 4D-Nucleofector™ System and choose the cell-type specific program for the human embryonic stem cell line H9, the cuvette size, P3 primary solution and the pulse CB-156 or CB-150 (see Note 12).
?. [Nucleofection of iPSCs]
Dissociate the cells to be nucleofected using TrypLE, centrifuge () and resuspend in mTeSR™1 with ROCKi.
Centrifuge: 400 34, 4 min
?. [Nucleofection of iPSCs]
Quickly put the cuvette(s) into the Nucleofector™ and press the start button to apply the pulse CB-156 or CB-150.
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The next day, wash the cells with  and change the medium to mTeSR™1 w/o ROCKi. Change the medium every day until next passaging (see Fig. 2b). Starting  after nucleofection, select the cells with an integrated construct with the appropriate antibiotic (see Note 14).
[PBS w/o Ca2+ and Mg2+]
?. [Nucleofection of iPSCs]
In order to determine the number of the integrated piggyBac constructs, use the piggyBac copy number kit from System Biosciences (see Note 15). To prepare genomic DNA, seed the cells in a 12-well plate (see Note 16). When confluent, wash once with  and add  to each well.
[PBS w/o Ca2+ and Mg2+]
[lysis buffer]
?. [Nucleofection of iPSCs]
Prepare two master mixes of , , and  per sample (one master mix with UCR1 primers for genomic DNA detection and one with PBcopy primers for piggyBac detection).
[ddH2O]
[SYBR® Green]
[primers]
?. [Nucleofection of iPSCs]
Calculate the copy number as follows [11]: ΔΔCt = 2(average PBcopy Ct–average UCR1 Ct), divide the ΔΔCt by 2 as there are two copies of the UCR1 sequence per genome.
?. [Nucleofection of iPSCs]
Determine the cell number, transfer 800,000 cells for each nucleofection into a 1.5 ml tube and centrifuge ().
Centrifuge: 400 34, 4 min
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Aspirate the supernatant and resuspend the cells in , mix with the DNA and transfer into an electroporation cuvette and close the lid. Avoid air bubbles while pipetting. Gently tap the cuvette to make sure that the sample covers the bottom.
[room temperature Nucleofector™solution with supplement]
?. [Nucleofection of iPSCs]
Immediately after, carefully remove the samples, add mTeSR™1 with ROCKi into the cuvette, mix by gently pipetting up and down two to three times and transfer the complete solution onto a Matrigel-coated plate with prewarmed medium and place in the incubator (see Note 13).
?. [Nucleofection of iPSCs]
Freeze the cells at  and thaw the plate at  to ensure complete cellular lysis.
-80 °C
0 Room temperature
?. [Nucleofection of iPSCs]
Detach the cells by pipetting up and down, transfer the lysates to 1.5 ml tubes and heat them at  for .
95 °C
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Centrifuge at  and transfer the supernatant to a new 1.5 ml tube. The lysates should be placed  if used immediately or stored at .
Centrifuge: 17000 34, 2 min
on ice
-20 °C
?. [Nucleofection of iPSCs]
Aliquot  per well of a 96-well plate and add  (≤). Seal the plate, carefully, mix by vortexing and briefly spin down.
[master mix]
[lysate]
[DNA]
?. [Nucleofection of iPSCs]
Run the qPCR with the following program:  at , at , 40 cycles of  for  and  for , followed by  at ,  at , and  at  (see Note 17).
50 °C
95 °C
95 °C
60 °C
95 °C
60 °C
95 °C
?. [Nucleofection of iPSCs]
In a separate tube, mix  with  per nucleofection reaction and bring to . Prepare Matrigel-coated cell culture plates with the desired volume of mTeSR™1 medium with ROCKi and prewarm in the incubator (see Note 11).
[Nucleofector™ solution]
[supplement]
0 Room temperature