TITLE: PERTURB SEQ PROTOCOL FOR EARLY POST-MITOTIC DOPAMINERGIC NEURONS
AUTHORS: Renuka Ravi Gupta, n.farbehi, hendersa, Vikram Khurana, Gist Croft, Lorenz Studer, Joseph Powell

[DESCRIPTION]
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.

[STEPS]
SECTION: Day -1: Coating wells with Poly - L ornithine(PO)
1. Coat 500 ul per well in a 48-well plate with 15 ug/ml PO in DPBS.
SECTION: Day -1: Coating wells with Poly - L ornithine(PO)
14. Incubate the plate overnight at 37ºC with 5% CO2 and 20.9% O2.
SECTION: Day 0: Coating wells with Laminin and Fibronectin
15. Thaw Fibronectin and Laminin on ice.
SECTION: Day 0: Coating wells with Laminin and Fibronectin
16. 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. 
 

SECTION: Day 0: Coating wells with Laminin and Fibronectin
17. Aspirate DPBS and add 500 ul of 2ug/ml Fibronectin and 1ug/ml Laminin in cold DPBS.
SECTION: Day 1: Transduction of  hESC CRISPRi dCAS9 D25 midbrain dopaminergic neurons(mDA neurons) with Lentiviral CRISPRi library supernatant
18. hESC CRISPRi dCAS9 are differentiated to D25 according to the following protocol: 
 

SECTION: Day 1: Transduction of  hESC CRISPRi dCAS9 D25 midbrain dopaminergic neurons(mDA neurons) with Lentiviral CRISPRi library supernatant
19. At D25, the cells were sorted by MACs sorting to obtain pure population dopaminergic mDA neurons.
 

SECTION: Day 1: Transduction of  hESC CRISPRi dCAS9 D25 midbrain dopaminergic neurons(mDA neurons) with Lentiviral CRISPRi library supernatant
20. 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%. 
SECTION: Day 1: Transduction of  hESC CRISPRi dCAS9 D25 midbrain dopaminergic neurons(mDA neurons) with Lentiviral CRISPRi library supernatant
21. Aspirate the fibronectin/ laminin coating and proceed immediately to the next step.
SECTION: Day 1: Transduction of  hESC CRISPRi dCAS9 D25 midbrain dopaminergic neurons(mDA neurons) with Lentiviral CRISPRi library supernatant
22. Add 200 ul /well of the virus media cocktail with the cells from step iii.
SECTION: Day 1: Transduction of  hESC CRISPRi dCAS9 D25 midbrain dopaminergic neurons(mDA neurons) with Lentiviral CRISPRi library supernatant
23. To increase the transduction efficiency, centrifuge the plate at 300 g for 20 minutes at 25 C (Spinoculation method).
24. Incubate the cells at 37ºC with 5% CO2 and 20.9% O2 for 16-18 hours.
SECTION: Day 2: Replace media
25. Aspirate the viral supernatant media gently and immediately add maturation media.
SECTION: Day 2: Replace media
26. Return the plate back to the incubator.
SECTION: Day -1: Coating wells with Poly - L ornithine(PO)
2. Aspirate the spent media.
SECTION: Day -1: Coating wells with Poly - L ornithine(PO)
3. Wash the cells 10 times with DPBS to remove the viral particles from the mDA neurons.
SECTION: Day -1: Coating wells with Poly - L ornithine(PO)
4. 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.
SECTION: Day -1: Coating wells with Poly - L ornithine(PO)
5. Add 100 ul HBSS +papain and incubate the neurons for 45 mins in the incubator.
SECTION: Day -1: Coating wells with Poly - L ornithine(PO)
6. Note: Ideally the neurons should dissociate as single cells.
SECTION: Day -1: Coating wells with Poly - L ornithine(PO)
7. Neutralize the papain with maturation media and collect the cells into 1.5ml eppendorf tubes.
SECTION: Day -1: Coating wells with Poly - L ornithine(PO)
8. 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.
SECTION: Day -1: Coating wells with Poly - L ornithine(PO)
9. Centrifuge the cells at 300g for 5 minutes.
SECTION: Day -1: Coating wells with Poly - L ornithine(PO)
10. Aspirate the spent media gently without disturbing the pellet.
SECTION: Day -1: Coating wells with Poly - L ornithine(PO)
11. Resuspend the cells in 300 ul of FACs buffer.
SECTION: Day -1: Coating wells with Poly - L ornithine(PO)
12. Transfer the cells with the FACs buffer into FACs tubes.
SECTION: Day -1: Coating wells with Poly - L ornithine(PO)
13. Sort the neurons to obtain 10% - 30% BFP positive cells and collect a minimum of 20000 cells for capture.
SECTION: Day 4: FACs Sort
27. Aspirate the spent media.
SECTION: Day 4: FACs Sort
28. Wash the cells 10 times with DPBS to remove the viral particles from the mDA neurons.
 

SECTION: Day 4: FACs Sort
29. Add 100 ul HBSS +papain and incubate the neurons for 45 mins in the incubator.
 

SECTION: Day 4: FACs Sort
30. Neutralize the papain with maturation media and collect the cells into 1.5ml eppendorf tubes.
 

SECTION: Day 4: FACs Sort
31. Centrifuge the cells at 300g for 5 minutes.
SECTION: Day 4: FACs Sort
32. Aspirate the spent media gently without disturbing the pellet.
SECTION: Day 4: FACs Sort
33. Resuspend the cells in 300 ul of FACs buffer.
SECTION: Day 4: FACs Sort
34. Transfer the cells with the FACs buffer into FACs tubes.
SECTION: Day 4: FACs Sort
35. Sort the neurons to obtain 10% - 30% BFP positive cells and collect a minimum of 20000 cells for capture.
SECTION: Day 4: 10x Capture (Contd)
36. 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

SECTION: Day 4: 10x Capture (Contd)
37. Load a minimum of 12000 cells for the capture.
SECTION: Day 4: 10x Capture (Contd)
38. 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.
SECTION: Day 4: 10x Capture (Contd)
39. Detailed protocol for 10X capture can be found in this link: 
https://cdn.10xgenomics.com/image/upload/v1684188462/support-documents/CG000510_ChromiumNextGEMSingleCell5_v2_CRISPR_UserGuide_Rev_C.pdf