Mar 03, 2026

Public workspaceNgn2 differentiation protocol for iPSCs without Ngn2 transgene knock-in

DOI
https://dx.doi.org/10.17504/protocols.io.81wgbowpnlpk/v1
  • Dahlia Rohm1,
  • Joshua Black1,
  • Sean McCutcheon1,
  • Alejandro Barrera1,
  • Shante Berry1,
  • Daniel Morone1,
  • Xander Nuttle2,
  • Celine de Esch2,
  • Derek Tai2,
  • Michael Talkowski2,
  • Nahid Iglesias1,
  • Charles Gersbach1
  • 1Duke University;
  • 2Harvard University
  • Gersbach Lab
Andrea R Daniel
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.81wgbowpnlpk/v1
Protocol Citation: Dahlia Rohm, Joshua Black, Sean McCutcheon, Alejandro Barrera, Shante Berry, Daniel Morone, Xander Nuttle, Celine de Esch, Derek Tai, Michael Talkowski, Nahid Iglesias, Charles Gersbach 2026. Ngn2 differentiation protocol for iPSCs without Ngn2 transgene knock-in. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbowpnlpk/v1
Manuscript citation:
 
Activation of the imprinted Prader-Willi syndrome locus by CRISPR-based epigenome editing.Rohm D, Black JB, McCutcheon SR, Barrera A, Berry SS, Morone DJ, Nuttle X, de Esch CE, Tai DJC, Talkowski ME, Iglesias N, Gersbach CA.Cell Genom. 2025 Feb 12;5(2):100770. doi: 10.1016/j.xgen.2025.100770.PMID: 39947136 
License: This is an open access protocol 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 protocol and it's working
Created: March 03, 2026
Last Modified: March 03, 2026
Protocol Integer ID: 244281
Keywords: neurons, iPSCs, differentiation, Ngn2, excitatory neurons from ipsc, ngn2 transgene knock, generating excitatory neuron, ngn2, transduction of virus, differentiation protocol for ipsc, ipsc, differentiation protocol
Funders Acknowledgements:
NIH
Grant ID: HG012053
Disclaimer
This protocol was adapted from work in the Yin Shen lab and the Li Gan lab and used by Dahlia Rohm in the Gersbach lab at Duke University.
Abstract
This protocol describes methods for generating excitatory neurons from iPSCs using transduction of virus expressing inducible neurogenin-2 (Ngn2).
Materials
ABC
ItemCat no.Supplier
ROCK Inhibitor Y-27632 72304 STEMCELL
Corning Matrigel growth-factor reduced basement membrane matrix, LDEV-free 354230Corning
AccutaseTM Cell detachment solution 07922 STEMCELL
MEM Non-Essential Amino Acids Solution (100X) (NEAA) 11140-050 Life Technologies
GlutaMAX Supplement (100X) 35050-061 Life Technologies
Recombinant Human/Murine/Rat BDNF 450-02 PeproTech
Recombinant Human NT-3 450-03 PeproTech
Laminin Mouse Protein, Natural 23017-015 Life Technologies
B-27 Supplement (50X) 17504-044 Life Technologies
N-2 Supplement (100X) 17502-048 Life Technologies
DMEM/F-12, HEPES 11330-032 Life Technologies
KnockOut DMEM/F-12 12660-012 Life Technologies
Neurobasal-A Medium 12349-015 Life Technologies
DPBS, no calcium, no magnesium 14190-144 Life Technologies
Poly-L-Ornithine P3655Sigma-Aldrich
Lentivirus – from lentiviral plasmid with reverse tetracycline transactivator (M2rtTA)20342 Addgene 
Lentivirus – made from lentiviral plasmid with Ngn2 under TetON promoter52047Addgene
Supplies
ABCD
Medium
ReagentsFinal concentrationVol for 100mL
Pre-differentiationMedium
KnockOut DMEM/F-12 100% 98 mL
N-2 Supplement (100X) 1X 1 mL
NEAA (100X) 1X 1 mL
BDNF 10 ng/mL Add before use
NT-3 10 ng/mL Add before use
Laminin 1 µg/mL Add before use
Doxycycline 2 µg/mL Add before use
Maturation Medium 
Neurobasal-A Medium 50% 48.5 mL
DMEM/F-12, HEPES 50% 48.5 mL
NEAA (100X) 1X 1 mL
B-27 Supplement (50X) 0.5X 1 mL
N-2 Supplement (100X) 0.5X 0.5 mL
GlutaMAX Supplement (100X) 0.5X 0.5 mL
BDNF 10 ng/mL Add before use
NT-3 10 ng/mL Add before use
Laminin1ug/mLAdd before use
Medium recipes
Troubleshooting
Preparation
Before beginning, make high-titre lentivirus from Addgene plasmids and concentrate to 50x of original concentration. Coat appropriate number of plates with Matrigel for iPSC culture.
Lentivirus – from lentiviral plasmid with reverse tetracycline transactivator (M2rtTA)20342 Addgene 
Lentivirus – made from lentiviral plasmid with Ngn2 under TetON promoter52047Addgene
Transduction of Ngn2 virus
Day 0: In 24-well plate, transduce 30,000 iPSCs (per well) with 2uL M2rtTA and 3uL TetON-Ngn2 lentiviruses (50x virus stock - 0.5x final) when plating cells with ROCKi. Can start with larger scale if necessary. 
Day 1: Media change with mTeSR Plus
Day 2 onwards: Expand and re-plate into 6-well plates or more.
Pre-differentiation protocol
Day -4: Plate 600k cells/well of a 12-well plate in mTeSR + ROCKi. This is 60% of the cell number needed for Day -3. (For a 6-well plate, plate 1.2 million cells/well).
Day -3 to day 0: Change media daily to pre-differentiation medium without ROCKi. 
Important notes: 
a. If cells are not confluent enough by day -3, can wait an extra day before initiating pre-differentiation.
b. Sometimes, cells start dying and looking unhappy in the last few days of pre-differentiation. In that case, adding an extra day to pre-differentiation can help increase cell number.
Post-differentation
Day 0:
a. Wash the Poly-L-Ornithine-coated plates three times using DPBS. Air dry in a tissue culture hood until all the DPBS is evaporated.

b.Dissociate the pre-differentiated cells using Accutase. For example, using 6-well plates:
i. Add 0.5 mL Accutase to each well and incubate at 37°C until cells are dissociated into single cells (~5 minutes).
ii. Add DPBS and collect the cells (do not pipette at this stage).
iii. First, add 1 mL DPBS and collect the cells without pipetting.
iv. Add 1.5 mL DPBS to rinse the wells and collect all the cells.
v. Centrifuge for 5 minutes at 200 RCF at room temperature.
vi. Aspirate the supernatant.
vii. Resuspend in 1 mL Maturation Media and use a P1000 pipette to dissociate the pellet into single cells. Triturate at least 30X.
viii. Count the number of live cells using Trypan blue. At least 90% of the cells should be single cells.

c. Plate the cells using Maturation Medium with 2 µg/mL doxycycline using the following table:
Plate typeMedia volumeCell density
24-well plate 0.5 mL / well 50-100K / well
12-well plate 1 mL / well 400-450K / well
6-well plate 2 mL / well 1-1.2M / well
10-cm dish 15 mL / dish 6-8M / dish
15-cm dish 30 mL / dish 20-25M / dish
 
Day 7: Remove half the volume of media from each well or dish and add the same amount of fresh maturation medium without doxycycline.
Day 14: Remove half the volume of media from each well or dish and add twice the amount of fresh maturation medium without doxycycline. For example, using 12-well plates, first remove 0.5 mL then add 1mL for a total of 1.5 mL.
Day 21: Remove 1/3rd the volume of media from each well or dish and add twice the amount of fresh maturation medium without doxycycline. At this point, the total volume of maturation medium should be double the initial volume. For example, using 12-well plates, there should be 2mL total.
After day 21: Change 1/3rd of the media using fresh maturation medium without doxycycline every week.
Harvest: Dissociating cells as single cells for live assays or fix/perm
Dissociation protocol adapted from Jerber et al. Nat Genet 2021 (ref. 1)
Wash cells with DPBS
Add Accutase with 20U/mL papain and incubate for 20-30 min
Add DMEM/F12 + ROCKi (DNAse optional, not used here) to quench, then transfer to tubes using P1000 (40um cell strainer optional)
Centrifuge 10 min at 200xg, then resuspend in PBS with 0.04% BSA for flow cytometry or downstream processing. For fix perm: spin down again and resuspend in fix/perm buffer.
Protocol references
1. Jerber, J., Seaton, D.D., Cuomo, A.S.E. et al. Population-scale single-cell RNA-seq profiling across dopaminergic neuron differentiation. Nat Genet 53, 304–312 (2021). https://doi.org/10.1038/s41588-021-00801-6