TITLE: Dissociation of neuronal culture to single cells for scRNA-seq (10x Genomics)  
AUTHORS: Julie Jerber, James Haldane, Juliette Steer, Daniel Pearce, Minal Patel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines a method for dissociating a human pluripotent stem cell-derived neuronal culture to single cells for loading onto a Chromium 10x chip for single cell RNA-sequencing. </div><div class = "text-block"><span>This protocol makes a distinction between </span><span style = "font-weight:bold;">early neuronal progenitors </span><span>and </span><span style = "font-weight:bold;">mature</span><span> neuronal cultures, as additional steps and reagents are required in order to sufficiently dissociate the latter. These include:</span></div><div class = "text-block"><span>- </span><a href="https://www.protocols.io/reagents/worthington-biochemical-corporation/dnase-vial-d2-513566" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">DNase Vial (D2)</span></a></div><div class = "text-block"><span>- </span><a href="https://www.protocols.io/reagents/worthington-biochemical-corporation/pds-kit-papain-vial-513567" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">PDS Kit Papain Vial</span></a></div><div class = "text-block"><span style = "font-style:italic;">Note: In our labs, iPS cells were undergoing a 52-day long differentiation process to dopaminergic neurons (adapted from </span><a href="https://doi.org/10.1038/nature10648" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">doi.org/10.1038/nature10648</span></a><span style = "font-style:italic;">), and were treated under the 'mature' conditions when harvested from day 20 onward, and as 'early neuronal progenitors' on day 11. Example images of neuronal culture from our labs can be found in the Guidelines of this protocol for reference.</span></div><div class = "text-block">This protocol assumes use of:</div><div class = "text-block"><span>- 12-well tissue culture plates (3.8 cm</span><span style = "vertical-align:super;">2</span><span> surface area per well ) for samples/wells being harvested for dissociation (see Materials).</span></div><div class = "text-block"><span>- A NucleoCounter</span><span style = "vertical-align:super;">®</span><span>NC-200™ for purposes of cell counting.</span></div></div>

[STEPS]
?. [Buffer Preparation]
Prepare Wash Buffer 2 by adding 10% BSA solution (100 μg/mL) to DPBS (-/-) to a final concentration of 400 μg/mL. i.e. add 60 μL 10% BSA solution to 15 mL of DPBS (-/-).
?. [Dissociation]
Aspirate the media from the well(s) of the neuronal culture and gently add  of DPBS (-/-) without disturbing the cell layer.
1 mL
?. [Dissociation]
Aspirate the DPBS (-/-) and add  of Dissociation Buffer to each well.
0.5 mL
?. [Dissociation]
Transfer cells to a   5% CO2 tissue culture incubator. Incubate for the appropriate time according to age of the culture:Early neuronal progenitors (~Day 11) - Young mature neurons (~Day 30) - Older mature neurons (~Day 52) - Check the progress of the dissociation after the indicated time. If necessary extend the incubation period for up to 10 mins more, checking the cells every few minutes.
37 °C
[(up to 20 mins)]
[(up to 30 mins)]
[(up to 35 mins)]
?. [Dissociation]
Following incubation inspect the cells under a microscope. The cells should be detaching from the plate and the cell layer should have a darkened appearance. Cells will have a rounded appearance as they dissociate and individual cells should be visible at the edges or in gaps in the cell layer.
?. [Dissociation]
Add  of Wash Buffer 1 per well.
2 mL
?. [Dissociation]
Use a P1000 pipette to repeatedly wash the buffer over the well(s) to detach the cells and dissociate them to a single-cell suspension. Sufficient dissociation is critical at this step. It is recommended to check the cell suspension under a microscope and if necessary pipette the cell suspension up and down further until the majority is single cells. Try to avoid over-pipetting the cells as this could affect viability.
?. [Dissociation]
Transfer the cell suspension into a 15mL centrifuge tube capped with a 40µm cell strainer.
?. [Dissociation]
Optional: If there are residual cells in the plate, wash well(s) with  of Wash Buffer 1 and transfer this suspension into the tube from the previous step.
0.5 mL
?. [Dissociation]
Centrifuge cells at .
Centrifuge: 150 34, 3 min
?. [Dissociation]
Aspirate supernatant and gently re-suspend the cell pellet in  of Wash Buffer 2 with a P1000 pipette.
1 mL
?. [Dissociation]
Transfer the cells to a Lo-bind 1.5mL Eppendorf tube.We recommend preparing and thawing necessary reagents for the 10x loading procedure during these next steps if you have not already done so.
?. [Dissociation]
Repeat steps 14 & 15 three times in a mini centrifuge (for a total of four centrifugations).
?. [Buffer Preparation]
Prepare Wash Buffer 1 (without DNase) by supplementing 15mL DMEM:F12 + GlutaMAX with ROCK inhibitor (Y-27632) to a final concentration of 10μM.Note: We make up 15mL of Wash Buffer 1 for every 6 wells of a 12 well plate to dissociate.For mature neurons, 1 vial DNase (D2) should be added for every 15mL of buffer prepared. However, DNase should only added immediately prior to use of the buffer when the cells are nearing the end of their dissociation. Keep the DNase at  until use.  ABCD1CellsDMEM/F12 + glutamaxRock inhibitor (Y-27632) 10mM stockDNase (D2)2Neuronal progenitors (~Day 11)15 mL15 uLNot required3Mature Neurons (> Day 20)15 mL15 uL1 vial (250uL)
4 °C
ABCD1CellsDMEM/F12 + glutamaxRock inhibitor (Y-27632) 10mM stockDNase (D2)2Neuronal progenitors (~Day 11)15 mL15 uLNot required3Mature Neurons (> Day 20)15 mL15 uL1 vial (250uL)
?. [Buffer Preparation]
Prepare Dissociation Buffer for mature neurons by combining Accutase with DPBS (-/-) at a 1:1 ratio. Add 1 vial Papain (PDS Kit) per 5mL buffer. Reconstitute the lyophilised papain with the Accutase/DPBS solution, replace the lid on the vial and invert several times ensuring all powder in the vial and on the lid is dissolved. Transfer solution back into the Dissociation Buffer tube. Dissociation Buffer for neuronal progenitors is undiluted Accutase only.  ABCD1CellsAccutaseDPBS (-/-)Papain (PDS kit)2Neuronal progenitors (~Day 11)0.5 mL per wellNot requiredNot required3Mature Neurons (> Day 20)2.5 mL2.5 mL1 vial
ABCD1CellsAccutaseDPBS (-/-)Papain (PDS kit)2Neuronal progenitors (~Day 11)0.5 mL per wellNot requiredNot required3Mature Neurons (> Day 20)2.5 mL2.5 mL1 vial
?. [Dissociation]
Optional: Test for ease of dissociation by gently pipetting ~ of the Dissociation Buffer against the cells with a P1000 pipette. If cells do not dissociate easily, extend digestion by  and repeat this step.
100 µl
?. [Cell Counts]
Note: This step describes a 1 in 10 dilution for cell counting on a NucleoCounter®NC-200™. Depending on method of cell counting and number of cells, this dilution step may not be necessary.Prepare a cell count sample tube for each suspension prepared by adding  of Wash Buffer 2 to a fresh Lo-bind 1.5ml Eppendorf tube.Take a  aliquot of the well-mixed cell suspension and add this to the  of Wash Buffer 2 in the prepared counting tube(s).
450 µl
50 µl
450 µl
?. [Cell Counts]
Count the cell suspension(s) on a NucleoCounter®NC-200™ and record % cell viability and viable cell concentration. Ensure less than 3% of cells are in aggregates.If >3% of the cells are in aggregates, triturate the cell suspension by pipetting up and down with a P1000 pipette. Carry out a second cell count on a new sample. Alternatively, dilute the sample and pass through a cell strainer, then centrifuge and resuspend in 1mL Wash Buffer 2 before repeating the cell count.
?. [Cell Counts]
Place the cell suspension tube(s)   during the cell counts and following steps.
on ice
?. [Cell Counts]
Using the cell counts, calculate the dilution of the cell suspension(s) necessary to produce the desired cell number for loading for the 10x. Dilutions should be made using Wash Buffer 2, to a final volume of .
1 mL
?. [Cell Counts]
Keeping the cell suspensions , proceed with the sample preparation and loading steps described in the 10x Genomics single cell gene expression kit guidelines.
on ice
?. [Dissociation]
For dissociation of mature neurons complete the preparation of Wash Buffer 1 within the last 5 minutes of incubation of the cells. Retrieve the DNase (D2) vial(s) from  storage. Reconstitute the vial(s) by adding 250 μL Wash Buffer 1 to each vial to make a 2mg/mL solution. Make sure to reconstitute all the powder (check the lid). Avoid vigorous mixing - do not vortex. Transfer the entire contents of the reconstituted vial(s) back into the Wash Buffer 1 tube. Use 250µL (1 vial) for every 15 mL buffer.
4 °C
?. [Cell Counts]
We recommend performing a post-10x loading cell count on the diluted cell suspension. This cell count can normally be performed without further dilution (depending on the method of cell counting). This cell count will confirm the cell number loaded for 10x and check the cell viability has not dropped significantly since the first cell count. It can also check the percentage cell aggregates has remained acceptable.