TITLE: Protocol for harvesting and dissociating mouse brain neurons  for single cell RNA Sequencing on the 10X Genomics platform
AUTHORS: Viktor Feketa, Elena O. Gracheva

[DESCRIPTION]
Single-cell RNA sequencing has emerged as a powerful method to characterize gene expression  on a single cell level. Producing useful data with this method critically relies on obtaining a  suspension of dissociated cells with high concentration and viability from the tissue of interest.  This protocol allows to isolate and dissociate mouse brain cellsinto a concentrated cell suspension  that is compatible with the 10X Genomics library preparation and sequencing pipeline and  enables capturing up to 10,000 single cells.

[GUIDELINES]
Isolation of neurons is based on the following published protocol with minor modifications: 

Vazirani, R. P., Fioramonti, X., Routh, V. H. Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing. J. Vis. Exp. (81), e50861, doi:10.3791/50861 (2013).
https://www.jove.com/t/50861/membrane-potential-dye-imaging-ventromedial-hypothalamusneurons-from

The protocol was further adapted for 10X Genomics platform according to the 10X Genomics "Cell Preparation Guide" (CG00053 Rev C):

https://support.10xgenomics.com/single-cell-gene-expression/sample-prep/doc/demonstratedprotocol-single-cell-protocols-cell-preparation-guide

References:

1. Vazirani, R. P., Fioramonti, X., Routh, V. H. Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing. J. Vis. Exp. (81), e50861, doi:10.3791/50861 (2013). 

2. https://support.10xgenomics.com/single-cell-gene-expression/sample-prep/doc/demonstratedprotocol-single-cell-protocols-cell-preparation-guide

[STEPS]
SECTION: Part 1: Advance preparation of solutions
1. Prepare stock solutions of media supplements
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1.1. Lactic acid: prepare 1 Molarity (M) (90 mg/mL) solution of lactic acid in nuclease-free water and  aliquot in50 µL aliquots. Store at -20 °C.

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1.2. GlutaMAX: aliquot original GlutaMAX (200 millimolar (mM)) solution into 100 µL aliquots. Store at-20 °C.

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1.3. B27 (minus insulin) supplement: thaw original solution and aliquot into 700 µL aliquots.  Store at -20 °C.

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2. Prepare 1 L of Brain Perfusion Solution (composition: 2.5 millimolar (mM) KCl, 7 millimolar (mM) MgCl2, 1.25 millimolar (mM)  NaH2PO4, 28 millimolar (mM) NaHCO3, 0.5 millimolar (mM) CaCl2, 7 millimolar (mM)glucose, 1 millimolar (mM)ascorbate, and 3 millimolar (mM) pyruvate  in nuclease-free water). Adjust the osmolarity to ~300 mOsm using approximately 67 g/L sucrose.  Oxygenate by bubbling with 95% O2 /5% CO2 for 15 minand adjust thepH 7.4. Filter with a  filter unit. Aliquot in 200 mL aliquots (each experiment will require approximately 200 mL of  perfusion solution). Aliquots can be stored at-20 °C for up to 2 months. The day before  experiment: thaw an aliquot of the Brain Perfusion Solution at4 °C15 min.

 We prepare the solution from the following specific components (for 1 L solution): 
      #  Component  Final Conc. (mM)  MW (g/mol)  Solid weight (mg/1000 mL)    1  Sucrose   96  342.3  67000    2  KCl  2.5  74.55  186.4    3  NaHCO3  28  84.01  2352    4  NaH2PO4 * H20  1.25  137.99  172.5    5  Glucose  7  180.16  1261    6  Sodium Ascorbate  1  198.11  198.1    7  CaCl2 * 2H20  0.5  147.01  73.5 (or 0.5ml of 1 M stock)    8  MgCl2  7  95.21  666.5 (or 7ml of 1 M stock)    9  Sodium Pyruvate  3  110.04  30 ml of 100 mM stock  
 

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3. The day before experiment, prepare a fresh 30 mL aliquot of Hibernate A media with 50 millimolar (mM)  glucose, osmolarity of280 mOsm, and 100 U/ml penicillin-streptomycin.
 





SECTION: Part 1: Advance preparation of solutions
3.1. In the sterile culture hood, transfer 30 mL of stock Hibernate A (pre-made by the supplier with 2.5 millimolar (mM) glucose and nominal osmolarity of275 mOsm) to the 50 mL culture flask.

SECTION: Part 1: Advance preparation of solutions
3.2.  Add 300 µL of 100X Pen-strep stock solution to 30 mL Hibernate A aliquot.

SECTION: Part 1: Advance preparation of solutions
3.3. Add NaCl (cell culture grade) to adjust osmolarity to 280 mOsm (~0 mg to 5 mg).

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3.4.  Store media aliquot at 4 °C in dark until the day of experiment.

SECTION: Part 1: Advance preparation of solutions
4. Fire-polish the tips of eight 9-inch glass Pasteur pipettes: 4 pipettes barely polished until the tips are no longer sharp (for cell transfers) and 4 pipettes for trituration with the following approximate tip opening diameters: 0.9 mm; 0.7 mm; 0.5 mm; 0.3 mm. The largest should be barely polished and the smallest should be about a third of that diameter.
SECTION: Part 2. Experimental procedure on the day of experiment.
5. Maintain RNAse-free conditions throughout the procedure. Wipe down all surfaces and tools with the "RNAse-away" reagent.
SECTION: Part 2. Experimental procedure on the day of experiment.
6. Thaw frozen aliquots of lactic acid, GlutaMax and B27 (minus insulin) supplements, vortex.
SECTION: Part 2. Experimental procedure on the day of experiment.
7. Prepare working solution of the Hibernate A media by adding the supplements for the following final working concentrations:
SECTION: Part 2. Experimental procedure on the day of experiment.
7.1. Lactic acid: 1 millimolar (mM) (add 30 µL of 1 Molarity (M) stock solution to the30 mL media aliquot).

SECTION: Part 2. Experimental procedure on the day of experiment.
7.2. GlutaMax: 0.5 millimolar (mM) (add 75 µLof 200 millimolar (mM) stck solution to the 30 mL media aliquot) .

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7.3. B27: 2% (add 600 µL of the stock solution to the 30 mL media aliquot).

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8. Mix media aliquot by inversion. Adjust pH to pH 7.4using 1 Normality (N) NaOH.

SECTION: Part 2. Experimental procedure on the day of experiment.
9. Oxygenate the thawed 200 mLaliquot of the Brain Perfusion Solution with 95% O2/5% CO2 on icefor at least 15 min.

SECTION: Part 2. Experimental procedure on the day of experiment.
10. Prepare a 50 mL culture flask labeled "digestion" and 9 polystyrene 50 mL conical tubes labeled "harvest", "dissection", "papain", "DNAse", "BSA", "BSA filtered", "BSA centrifugation", "trituration", "cell suspension".

SECTION: Part 2. Experimental procedure on the day of experiment.
11. Distribute Hibernate-A media: transfer2 mL to the "harvest" tube,9 mLto "dissection" tube, 4 mL to "papain" tube, 5.5 mL to "DNAse" tube; 5 mL to "trituration" tube. Preserve the rest of Hibernate A media for next steps.

SECTION: Part 2. Experimental procedure on the day of experiment.
12. Place "harvest" and "dissection" tubes with media on ice near the animal dissection area.

SECTION: Part 2. Experimental procedure on the day of experiment.
13. Prepare RNA-se free area for animal dissection: wipe all surfaces and tools with "RNAse Away" reagent.
SECTION: Part 2. Experimental procedure on the day of experiment.
14. Prepare vibratome: get a new blade, wash with 70% ethanol followed by ultrapure water, load the blade in the vibratome blade holder.
SECTION: Part 2. Experimental procedure on the day of experiment.
15. Prepare area and tools for cardiac perfusion and animal dissection: bath for cardiac perfusion with absorbent pad, surgical tools, cardiac perfusion system with an oxygenation line inserted into perfusion reservoir.
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16. Fill cardiac perfusion system with ultrapure water and let it run through to clean.
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17. Add isoflurane to induction chamber for animal anesthesia.
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18. Fill outer vibratome bath with ice and install on the vibratome.
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19. Place 2 glass Petri dishes on ice.

SECTION: Part 2. Experimental procedure on the day of experiment.
20. Prepare oxygenation line (with 95% O2/5% CO2) to be later inserted into inner vibratome bath.
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21. Place a mouse into induction chamber with isoflurane and wait until it stops breathing.
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22. Take mouse out and verify depth of anesthesia by absence of response to toe pinch.
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23. Pour ~30 mL of the ice-cold oxygenated Brain Perfusion Solution into reservoir of cardiac perfusion system just prior to dissection, fill tubing, and stop when about 20 mL of solution is left in the reservoir to be used for perfusion. Continue oxygenating the rest of the Brain Perfusion Solutionon ice.

SECTION: Part 2. Experimental procedure on the day of experiment.
24. Perform mouse dissection. 
 
Open abdominal cavity with scissors and peel back skin above ribcage, exposing diaphragm. Cut diaphragm and rib cage towards the forelimbs on both sides, then cut diaphragm along the edge of the rib cage to expose thoracic cavity. Be careful not to puncture the heart or big vessels. Peel back the ribcage towards the head, exposing the heart.
SECTION: Part 2. Experimental procedure on the day of experiment.
25. Insert the cardiac perfusion needle into left ventricle. Cut the right atrium with scissors.
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26. Start the flow of perfusion system.
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27. Perfuse about 15 mL of perfusion solution (~1 min) until the effluent is clear. Success of perfusion can be assessed by internal organs changing color to a lighter shade.

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28. Fill a petri dish on ice with ~ 15 mL of Brain Perfusion Solution.

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29. Decapitate the mouse, Extract brain:
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30. Make a coronal cut between cerebellum and the rest of the brain, trying to make it as flat and perpendicular to the rostro-caudal axis of the brain as possible.
 

SECTION: Part 2. Experimental procedure on the day of experiment.
31. Lift the brain from the solution with a curved spatula and gently dab with filter paper to dry, especially the flat coronal aspect which will be glued down to the vibratome stage.
SECTION: Part 2. Experimental procedure on the day of experiment.
32. Put a little drop of super glue on the vibratome stage and spread it with a cotton tip to an area slightly larger than the brain.
SECTION: Part 2. Experimental procedure on the day of experiment.
33. Put the brain down on the stage area covered with glue, coronal aspect (now being the caudal end) down, rostral end up.
SECTION: Part 2. Experimental procedure on the day of experiment.
34. Mount the vibratome stage with the glued brain in the inner vibratome bath. About 10 s after the brain attachment, fill the vibratome bath with the remaining oxygenated Brain Perfusion Solution to completely cover the brain in the bath.

SECTION: Part 2. Experimental procedure on the day of experiment.
35. Rotate the stage of the vibratome with the brain to orient ventral (hypothalamic side) towards and dorsal cortex away from the blade.
SECTION: Part 2. Experimental procedure on the day of experiment.
36. Make sure the oxygenation line is inserted into vibratome bath and is turned on throughout the brain slicing.
SECTION: Part 2. Experimental procedure on the day of experiment.
37. Lower the vibratome blade into cutting position.
SECTION: Part 2. Experimental procedure on the day of experiment.
38. Using vibratome control pad, move the stage and blade as needed to perform the first cut close to the rostral end of the brain (facing up in the bath).
SECTION: Part 2. Experimental procedure on the day of experiment.
39. Start cutting   thick slices (vibratome settings: speed: 0.2 mm/sec, amplitude: 1 mm), observing the anatomical cues until the target area is reached. The level of bregma+1.0mm is reached approximately when left and right parts of corpus callosum meet in the middle.

SECTION: Part 2. Experimental procedure on the day of experiment.
40. After reaching the target area, cut two consecutive    thick slices to be collected.
SECTION: Part 2. Experimental procedure on the day of experiment.
41. Pour all 9 mL of Hibernate A media from the "dissection" tube into the second empty Petri dish on ice.

SECTION: Part 2. Experimental procedure on the day of experiment.
42. Transfer two target slices with a spatula from the vibratome bath into Petri dish with Hibernate A media.
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43. Using 27G needles attached to 1 mL syringes used as cutting tools, dissect the target brain areas from slices. Further cut dissected pieces in two, to produce tissue pieces about 1x1 mm in size.

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44. Collect tissue pieces with a glass pipette, transfer them to the "harvest" tube with Hibernate A media, and place the tube on ice.

SECTION: Part 2. Experimental procedure on the day of experiment.
45. Prepare digestion solution: add 80 U of stock papain suspension to the "papain" tube with 4 mL of media (for a final concentration of 20 U/ml; calculate the volume of papain to get80 U beforehand based on the activity of specific batch, usually 65 µLto 85 µL). Mix by inversion and place in 34 °C water bath. Check and mix by inversion every minute until the media is no longer cloudy (~4 min).

SECTION: Part 2. Experimental procedure on the day of experiment.
46. During "papain" incubation, place the "harvest" tube with tissue pieces into 34 °C water bath.

SECTION: Part 2. Experimental procedure on the day of experiment.
47. Using 0.22-µm syringe filter, filter papain solution into a "digestion" culture flask.
SECTION: Part 2. Experimental procedure on the day of experiment.
48. Using glass pipette, transfer tissue pieces from "harvest" tube into "digestion" culture flask with papain. Shake "digestion" flask to make sure tissue pieces are not clumped together but are floating separately to ensure proper digestion.
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49. Incubate "digestion" flask in a shaking water bath at 34 °C with shaking at  .

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50. Meanwhile, prepare media solutions for trituration:
SECTION: Part 2. Experimental procedure on the day of experiment.
50.1. Prepare DNAse solution: add 500 µL of Hibernate A media to a vial with DNAse solid, gently  but thoroughly mix to dissolve completely (do not vortex, DNAse is sensitive to shear).  Transfer 500 µL of dissolved DNAse to the "DNAse" tube with 5.5 mLof Hibernate A media  (final volume 6 mL, final concentration of DNAse0.1 mg/mL). Invert gently to mix (do not  vortex).

SECTION: Part 2. Experimental procedure on the day of experiment.
50.2. Prepare BSA solution: weigh 160 mg of bovine serum albumin solid and add to the "bsa" tube with 2 ml of Hibernate A (final concentration: 8% BSA). Vortex for 30 sec. Using 0.22 µm syringe filter, filter BSA solution into another "bsa filtered" tube. Transfer 1 mL of filtered BSA to the "bsa centrifugation" tube.

SECTION: Part 2. Experimental procedure on the day of experiment.
50.3. Place 30 μm Miltenyi SmartStrainer onto "cell suspension" tube to collect and filter cell  suspension.

SECTION: Part 2. Experimental procedure on the day of experiment.
51. When 30 min digestion is done, transfer tissue pieces from "digestion" to the "trituration" tube using glass pipette. Invert once and let tissue pieces settle. Aspirate almost all media with glass pipette, leaving only tissue pieces.

SECTION: Part 2. Experimental procedure on the day of experiment.
52. Add 3 mL of the trituration media ("DNAse" tube) to the "trituration" tube with tissue pieces. Triturate with the largest pipette (0.9 mm) 10 times (draw large volume to collect all tissue pieces with each trituration stroke) over approximately 30 s. Wait 4 min for tissue pieces to settle. Use the second-largest (0.7 mm tip opening) glass pipette to transfer 2 mL from the top of the cell suspension onto the cell strainer on top of "cell suspension" tube.

SECTION: Part 2. Experimental procedure on the day of experiment.
53. Add 2 mL of trituration media to the "trituration" tube. Triturate 10 times with the second-largest 0.7 mm pipette. Wait 3 min for pieces to settle. Use the third (0.5mm) glass pipette to transfer the top 2 mL to the cell strainer/"cell suspension" tube.

SECTION: Part 2. Experimental procedure on the day of experiment.
54. Add 1 mLof trituration media to the "trituration" tube. Triturate 5 times with the third 0.5 mm pipette. Wait 2 minfor tissue pieces to settle. Use the fourth 0.3 mm glass pipette to transfer all the remaining solution from the "trituration" to the cell strainer/"cell suspension" tube.

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55. Remove cell strainer from the "cell suspension" tube and layer all the solution from this tube onto the BSA layer in the "BSA centrifugation" tube using a glass pipette. 
 

SECTION: Part 2. Experimental procedure on the day of experiment.
56. Centrifuge the "BSA centrifugation" tube1000 rpm, 5 min. Using an Eppendorf 5810R swinging bucket centrifuge, this corresponds to 67 rcf. Set "break" setting to '0', i.e. no breaking.

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57. After centrifugation is done, carefully aspirate almost all media from the tube, leaving about 50 µL of solution above the cell pellet.

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58. Add 950 µL of Hibernate A media to cell pellet. Resuspend cells with the glass pipette (gently pipette up and down 10 times).

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59. Transfer all (~1 mL) of the cell suspension from the "bsa centrifugation" tube to a new 2 mLEppendorf LoBind tube.

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60. Centrifuge at 300 rcf, 5 min in a small tabletop centrifuge (Eppendorf 5424: 1787 rpm) at 34 Room temperature.

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61. Carefully remove supernatant to leave ~50 µLof the solution, avoiding disrupting the cell pellet (it won't be visible) and creating bubbles.

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62. Using glass pipette, carefully resuspend the cell pellet in the remaining ~50 µL of solution (pipette up and down about 10 times, avoid creating bubbles). This is the final cell suspension used for 10X library preparation. Place and store tube on ice until starting the 10X single cell protocol.

SECTION: Part 2. Experimental procedure on the day of experiment.
63. To determine the volume of solution to use for library preparation, determine cell concentration by counting cells using a hemocytometer. Transfer a 10 µLaliquot from the final cell suspension to a new2 mL LoBind tube. Add 10 µLof Trypan Blue stain. Mix gently with a pipette. Pipette 10 µL into a hemocytometer chamber. Count the number of live (transparent) and dead (blue) cells under the microscope. In case of harvesting primary and secondary motor areas from two   thick slices (4 tissue pieces ~1x2 mm in size), the expected cell concentration is ~1,000-2,000 cells/µl, expected viability is ~85%. To recover 10,000 cells in 10X protocol, the optimal concentration is 700-1,200 cells/µl. If obtained cell concentration is much higher than that, dilute cell suspension to the desired concentration by adding appropriate volume of Hibernate A media.

SECTION: Part 2. Experimental procedure on the day of experiment.
64. Proceed with the 10x Genomics Single Cell Protocol.
 

SECTION: Expected results
65. In case of harvesting primary and secondary motor cortex areas from two   thick slices (4 tissue  pieces ~1x2 mm in size), the expected cell concentration is ~1,000-2,000 cells/µl, expected viability is  ~85%.

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66. Figure 1 shows a representative image of the cell suspension loaded in hemocytometer for counting.
 
 

 
https://support.10xgenomics.com/single-cell-gene-expression/sample-prep/doc/demonstrated-protocol-single-cell-protocols-cell-preparation-guide
SECTION: Part 2. Experimental procedure on the day of experiment.
29.1. Cut skin above skull from caudal to rostral end and peel away to expose skull.
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29.2. Make a midline cut with scissors in the skull towards the eye sockets. Use fine Graefe forceps to break and peel pieces of skull away from midline, exposing the brain.
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29.3. Make a coronal cut with scissors between olfactory bulbs and the rest of the brain, and another cut between the brain and spinal cord to mobilize the brain.
SECTION: Part 2. Experimental procedure on the day of experiment.
29.4. Use a spatula to slightly lift the brain from the skull, use scissors to cut optical tracts, and finally extract the mobilized brain from the skull with a spatula (hippocampal tool) and push it into the Petri dish with the Brain Perfusion Solution.