Feb 29, 2024

Public workspacePERTURB SEQ PROTOCOL FOR EARLY POST-MITOTIC DOPAMINERGIC NEURONS V.2

This protocol is a draft, published without a DOI.
  • 1Garvan Weizmann Center for Cellular Genomics, Garvan Institute of Medical Research, Sydney, NSW 2010, Australia;
  • 2Garvan Institute of Medical Research, Sydney, NSW 2010, Australia;
  • 3Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA;
  • 4School of Medical Science, University of New South Wales, Sydney, NSW, 2052, Australia;
  • 5Graduate School of Biomedical Engineering, University of New South Wales, Sydney, NSW, 2052, Australia;
  • 6The Centre for Stem Cell Biology, Developmental Biology Program, Sloan Kettering Institute for Cancer Research, New York, NY, USA;
  • 7Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115, USA;
  • 8The New York Stem Cell Foundation Research Institute, New York, NY, USA
Renuka Ravi Gupta
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Protocol Citation: Renuka Ravi Gupta, Nona Farbehi, hendersa, Vikram Khurana, Gist Croft, Lorenz Studer, Joseph Powell 2024. PERTURB SEQ PROTOCOL FOR EARLY POST-MITOTIC DOPAMINERGIC NEURONS. protocols.io https://protocols.io/view/perturb-seq-protocol-for-early-post-mitotic-dopami-c9wqz7dwVersion created by Renuka Ravi Gupta
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: In development
We are still developing and optimizing this protocol
Created: February 29, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 95920
Keywords: ASAPCRN, Perturb-Seq, CRISPRi, Lentiviral Transduction, Stem Cells, Dopaminergic Neurons, Parkinson Disease
Funders Acknowledgements:
ASAP
Grant ID: ASAP-000472
Abstract
We have developed a protocol where genetic perturbations via CRISPRi machinery are introduced into early post mitotic dopaminergic neurons through lentiviral transduction . The selected cells were used to generate sequencing ready single cell libraries to assess gene expression profiles and CRISPR mediated perturbations.
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Materials

ABC
MATERIALCOMPANYCATALOG
48 well TC treated plateFalcon353078
15ml polypropylene centrifuge tubesFalcon352096
5ml serological pipettesCorning4487
10ml serological pipettesCorning4488
DNA Low-bind tubes 1.5mlEppendorf022431021
P1000 tipNeptuneBT1250
FBSBovogen2008A
DPBSThermoFisher Scientific14040133
Neurobasal MediaThermoFisher Scientific21103049
B27 w/o vit AThermoFisher Scientific12587-010
L-glutamineThermoFisher ScientificL3000015
Pen-StrepThermoFisher Scientific12260
BDNF (Brain Derived Neurotrophic Factor)R&D248-BDB
GDNF (Glial Cell line Derived Neurotrophic Factor)Peprotech450-10
Ascorbic AcidSigma4034
cAMPSigmaD0627
TGF-B (Transforming Growth Factor - b)R&D243-B3
DAPT Tocris2634
Polyornithine (PO)SigmaP3655
Cultrex Mouse Laminin IR&D3400-010-1
FibronectinCorningFAL356008
REAGENT COMPOSITION

AB
MEDIA 2
REAGENTVOLUME IN ML
Neurobasal Media480
B27 without Vit A (10x)10
Pen-Strep5
L-Glutamine5

ABCD
MATURATION MEDIA (MM) (25ml)
REAGENTSTOCK SOLUTIONWORKING SOLUTIONVOLUME IN UL
Media 2 --24796
BDNF10 ug/ml20ng/ml50
GDNF10 ug/ml20 ng/ml50
AA100mM200uM50
cAMP100mM200uM50
DAPT100mM10uM2.5
TGF-B20 ug/ml1ng/ml1.25

AB
FACS BUFFER (PBS +2% FBS)
REAGENTVOLUME IN ML
PBS49
FBS1

Day -1: Coating wells with Poly - L ornithine(PO)
Day -1: Coating wells with Poly - L ornithine(PO)
Coat 500 ul per well in a 48-well plate with 15 ug/ml PO in DPBS.
Aspirate the spent media.
Wash the cells 10 times with DPBS to remove the viral particles from the mDA neurons.
Note: Ensure the robust adherence of neurons throughout the washing procedures. To mitigate the risk of neuronal detachment, it is imperative to conduct microscopic inspections during each wash step.
Add 100 ul HBSS +papain and incubate the neurons for 45 mins in the incubator.
Note: Ideally the neurons should dissociate as single cells.
Neutralize the papain with maturation media and collect the cells into 1.5ml eppendorf tubes.
Note: If the neurons are still present as a sheet or have clumps, use a P1000 tip, pipette the cells up and down to break them into single cell suspension.
Centrifuge the cells at 300g for 5 minutes.
Aspirate the spent media gently without disturbing the pellet.
Resuspend the cells in 300 ul of FACs buffer.
Transfer the cells with the FACs buffer into FACs tubes.
Sort the neurons to obtain 10% - 30% BFP positive cells and collect a minimum of 20000 cells for capture.
Incubate the plate overnight at 37ºC with 5% CO2 and 20.9% O2.
Day 0: Coating wells with Laminin and Fibronectin
Day 0: Coating wells with Laminin and Fibronectin
Thaw Fibronectin and Laminin on ice.
Aspirate 250ul of coated PO from each well of the 48 well plate and wash the wells with 1 ml of DPBS. Repeat two more times for a total of 3 x DPBS washes.
Note
Do not let the wells dry out.

Aspirate DPBS and add 500 ul of 2ug/ml Fibronectin and 1ug/ml Laminin in cold DPBS.
Day 1: Transduction of hESC CRISPRi dCAS9 D25 midbrain dopaminergic neurons(mDA neurons) with Lentiviral CRISPRi library supernatant
Day 1: Transduction of hESC CRISPRi dCAS9 D25 midbrain dopaminergic neurons(mDA neurons) with Lentiviral CRISPRi library supernatant
hESC CRISPRi dCAS9 are differentiated to D25 according to the following protocol:
CITATION
Tae Wan Kim, Jinghua Piao, So Yeon Koo, Sonja Kriks, Sun Young Chung, Doron Betel, Nicholas D. Socci, Se Joon Choi, Susan Zabierowski, Brittany N. Dubose, Ellen J. Hill, Eugene V. Mosharov, Stefan Irion, Mark J. Tomishima, Viviane Tabar, Lorenz Studer. Biphasic Activation of WNT Signaling Facilitates the Derivation of Midbrain Dopamine Neurons from hESCs for Translational Use. protocols.io.
LINK
https://protocols.io/view/biphasic-activation-of-wnt-signaling-facilitates-t-bu7znzp6

At D25, the cells were sorted by MACs sorting to obtain pure population dopaminergic mDA neurons.
CITATION
Tae Wan Kim. Dopamine neuron enrichment using MACS. protocols.io.
LINK
https://protocols.io/view/dopamine-neuron-enrichment-using-macs-cyrfxv3n

Prepare 15 ml tubes with 200k D25 pure population mDA neuronal suspension (CD49e neg) with concentrated lentiviral supernatants at an Multiplicity of Infection (MOI) of 10 - 30%.
Aspirate the fibronectin/ laminin coating and proceed immediately to the next step.
Add 200 ul /well of the virus media cocktail with the cells from step iii.
To increase the transduction efficiency, centrifuge the plate at 300 g for 20 minutes at 25 C (Spinoculation method).
Incubate the cells at 37ºC with 5% CO2 and 20.9% O2 for 16-18 hours.
Day 2: Replace media
Day 2: Replace media
Aspirate the viral supernatant media gently and immediately add maturation media.
Return the plate back to the incubator.
Day 4: FACs Sort
Day 4: FACs Sort
Aspirate the spent media.
Wash the cells 10 times with DPBS to remove the viral particles from the mDA neurons.
Note
Ensure the robust adherence of neurons throughout the washing procedures. To mitigate the risk of neuronal detachment, it is imperative to conduct microscopic inspections during each wash step.

Add 100 ul HBSS +papain and incubate the neurons for 45 mins in the incubator.
Note
Ideally the neurons should dissociate as single cells.

Neutralize the papain with maturation media and collect the cells into 1.5ml eppendorf tubes.
Note
If the neurons are still present as a sheet or have clumps, use a P1000 tip, pipette the cells up and down to break them into single cell suspension.

Centrifuge the cells at 300g for 5 minutes.
Aspirate the spent media gently without disturbing the pellet.
Resuspend the cells in 300 ul of FACs buffer.
Transfer the cells with the FACs buffer into FACs tubes.
Sort the neurons to obtain 10% - 30% BFP positive cells and collect a minimum of 20000 cells for capture.
Day 4: 10x Capture (Contd)
Day 4: 10x Capture (Contd)
Sequencing specifications are as follows:
For the Gene Expression library. The standard read depth is 30000 reads per cell. For the CRISPR library, the stand read depth is 7500 reads per cell. To calculate the number of cells needed for the capture, we use the following formula
Total number of cells = Number of genes x Number of guides / genes x Number of cells/ guide, where Number of genes =30 Number of guides/ genes = 2 (since we are using the dual guide strategy) Number of cells per guide = 200 Total number of cells =30 genes x 2 dual guides x 200 cells = 12000 cells
Load a minimum of 12000 cells for the capture.
The 10x capture was performed using the Chromium Next GEM Single Cell 5’ Reagent Kit v2 (Dual Index with Feature Barcode technology for CRISPR screening.
Protocol references
Citations
Step 18
Tae Wan Kim, Jinghua Piao, So Yeon Koo, Sonja Kriks, Sun Young Chung, Doron Betel, Nicholas D. Socci, Se Joon Choi, Susan Zabierowski, Brittany N. Dubose, Ellen J. Hill, Eugene V. Mosharov, Stefan Irion, Mark J. Tomishima, Viviane Tabar, Lorenz Studer. Biphasic Activation of WNT Signaling Facilitates the Derivation of Midbrain Dopamine Neurons from hESCs for Translational Use
https://protocols.io/view/biphasic-activation-of-wnt-signaling-facilitates-t-bu7znzp6
Step 19
Tae Wan Kim. Dopamine neuron enrichment using MACS
https://protocols.io/view/dopamine-neuron-enrichment-using-macs-cyrfxv3n