Jan 12, 2023

Public workspaceNucleofection (Amaxa) and electroporation (Biorad) of hPSCs V.2

DOI
dx.doi.org/10.17504/protocols.io.e6nvwkkewvmk/v2
  • 1University of California, Berkeley;
  • 2Albert Einstein College of Medicine
Devin E Snyder
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwkkewvmk/v2
External link: https://doi.org/10.7554/eLife.79208
Collection Citation: Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner 2023. Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwkkewvmk/v2Version created by Devin E Snyder
Manuscript citation:
Hanqin Li, Oriol Busquets, Yogendra Verma, Khaja Mohieddin Syed, Nitzan Kutnowski, Gabriella R Pangilinan, Luke A Gilbert, Helen S Bateup, Donald C Rio, Dirk Hockemeyer, Frank Soldner (2022) Highly efficient generation of isogenic pluripotent stem cell models using prime editing eLife 11:e79208

https://doi.org/10.7554/eLife.79208
License: This is an open access collection distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this collection and it's working
Created: January 12, 2023
Last Modified: May 31, 2024
Collection Integer ID: 75230
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000486
Abstract
This collection describes the standard procedure for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) into human pluripotent stem cells (hPSCs) using nucleofection or electroporation.

Collection Overview
Preparing MEF-cultured hPSCs for nucleofection
Preparing Feeder-free hPSCs for nucleofection
Preparing plasmids for nucleofection
Preparing mRNA for nucleofection
In vitro assembling of RNP for nucleofection
Nucleofection of hPSCs
Electroporation of hPSCs
A. Preparation of MEFs-cultured hPSCs for electroporation
B. Preparing plasmids for electroporation
C. Electroporation
FACS-based enrichment of transfected cells
A. Preparation of samples for FACS sorting
B. After FACS

General notes
  1. Throughout these protocols, 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.
Materials

ItemVendorCatalog #
DMEM/F12 Thermo Fisher 11320082
DPBS w/o calcium and magnesium Corning MT21031CV
Fetal Bovine Serum (FBS) Corning 35-011-CV
Knockout Serum Replacement Thermo Fisher 10828-028
L-Glutamine Sigma G8540
Penicillin & Streptomycin (100X) Thermo Fisher 15140163
MEM Non-Essential Amino Acids (100X) Thermo Fisher 11140050
Heat Stable Recombinant Human FGF2 Thermo Fisher PHG0360
Collagenase type IV Thermo Fisher 17104019
2-Mercaptoethanol Sigma M3148
mTeSR-plus STEMCELL Technologies 100-0276
StemFlex Thermo Fisher A3349401
Vitronectin (VTN-N) Recombinant Human Protein, Truncated Thermo Fisher A14700
Accutase Thermo Fisher SCR005
Dispase STEMCELL Technologies NC9995391
Y-27632 Chemdea CD0141
Cas9, purified protein, 40uM Macrolab, QB3 UC Berkeley
Synthetic pegRNAs IDT or Synthego
Synthetic sgRNAs Synthego
P3 primary Cell 4D X kit S Lonza V4XP-3032
Countess™ Cell Counting Chamber Slides Thermo Fisher C10228
pCMV-PE2 Addgene 132775
4D-Nucleofector TM Core + X Unit Lonza AAF-1002B, AAF-1002X
5 ml polystyrene round-bottom tube with cell-strainer cap Corning 352235
Cell-strainer (70 µm) Fisher 07201431
Gene Pulser Xcell Eukaryotic System Bio-Rad 1652661
Gene Pulser Electroporation Cuvettes, 0.4 cm gap Bio-Rad 1652081
Exact N Amp Blood PCR Kit SigmaXNAB2-1TK

Files
Protocol
Icon representing the file Preparing MEF-cultured hPSCs for nucleofection
Name
Preparing MEF-cultured hPSCs for nucleofection
Version 2
, University of California, Berkeley
Ezgi BoothUniversity of California, Berkeley
Protocol
Icon representing the file Preparing feeder-free hPSCs for nucleofection
Name
Preparing feeder-free hPSCs for nucleofection
Version 1
, University of California, Berkeley
Ezgi BoothUniversity of California, Berkeley
Protocol
Icon representing the file Preparing plasmids for nucleofection of hPSCs
Name
Preparing plasmids for nucleofection of hPSCs
Version 1
, University of California, Berkeley
Ezgi BoothUniversity of California, Berkeley
Protocol
Icon representing the file Preparing mRNA for nucleofection of hPSCs
Name
Preparing mRNA for nucleofection of hPSCs
Version 1
, University of California, Berkeley
Ezgi BoothUniversity of California, Berkeley
Protocol
Icon representing the file In vitro assembling of RNP for nucleofection of hPSCs
Name
In vitro assembling of RNP for nucleofection of hPSCs
Version 1
, University of California, Berkeley
Ezgi BoothUniversity of California, Berkeley
Protocol
Icon representing the file Nucleofection of hPSCs
Name
Nucleofection of hPSCs
Version 1
, University of California, Berkeley
Ezgi BoothUniversity of California, Berkeley
Protocol
Icon representing the file Electroporation of hPSCs
Name
Electroporation of hPSCs
Version 1
, University of California, Berkeley
Ezgi BoothUniversity of California, Berkeley
Protocol
Icon representing the file FACS-based enrichment of transfected hPSCs
Name
FACS-based enrichment of transfected hPSCs
Version 1
, University of California, Berkeley
Ezgi BoothUniversity of California, Berkeley