Single-cell dissociation and RNA sequencing

Hb dissociation protocol

Introduction

Protocol for dissecting and dissociating medial prefrontal cortex (mPFC), motor cortex (MCtx) and habenula (Hb) for inDrops sequencing. Separating left and right hemispheres. Protocol requires a team of 4 people for the dissection steps. 

Materials

• Choline solution - per 2 L stock
  • 2 L H₂O
  • 4.2 g NaHCO₃
  • 4.32 g glucose
  • 0.345 g NaH₂PO₄.H₂O
  • 15 mL MgCl₂.6H₂O (1M stock)
  • 5 mL KCl (1M stock)
  • 20 mL HEPES (pH 7.0 - 7.2, 1M stock)
• Choline solution - per 500 mL (make total of 2 L for 6-8 animals)
  • 500 mL choline stock solution, bubbled
  • 7.68 g choline chloride
  • 1.15 g ascorbic acid
  • 0.17 g pyruvic acid
  • pH 7.3 - 7.5
• Choline perfusion & microdissection solution - 100 mL
  • Choline solution (see above)
  • TTX 1:1000
  • AP-V 1:1000 or CPP 1:1000
• Dissociation Media (DM) - 500 mL stock 
  • 500 mL HBSS (ThermoFisher Scientific Cat No. 14170112)
  • 5 mL 1M HEPES
  • 86 mg kynurenic acid (dissolve in 1 mL 0.5M NaOH and add full volume)
  • 0.43 g MgCl₂.6H₂O
  • 3.15 g D-glucose
  • Bubble with 95% O₂, 5% CO₂ and adjust pH to 7.35 (add approx. 5 mL 1M NaOH)
  • Sterilize under hood with 0.22 filter
• Papain 2x (P) - 1 vial per tube, prepare fresh (x6 for 3 regions x 2 hemispheres) 
  • Papain from Worthington kit
  • PS (prewarmed)
  • DNAse I (1 vial)
  • Dilute papain in 3.25 mL DM (sterile, prewarmed). Final papain concentration: 40 U/mL.
  • Dilute 1 vial of DNAse I (2 mg/mL) with 300 µL DM
  • Add 200 µL of DNAse I in PS solution to Papain solution
  • TTX 1:1000
  • AP-V 1:1000 or CPP 1:1000
  • Keep at RT
• Trypsin inhibitor (TI) - make 2 (total 45 mL: 7.5 mL per tube, x6 for 3 regions x 2 hemispheres)
  • Resuspend a new vial of TI with 32 mL oxygenated DM
  • Take 28 mL aliquot
  • Add O₂/CO₂ to displace air
  • Keep on ice
• TI 1:10 (Quench Buffer) - make 2 (total 36 mL: 6 mL per tube, x6 for 3 regions x 2 hemispheres)
  • 22 mL DM
  • 2500 µL TI
  • 300 µL DNAse I
  • Keep on ice
• DM+BSA - divide into 2 portions (total 55.2 mL: 3.2 mL + 9 mL per tube, x6 for 3 regions x 2 hemispheres)
  • 65 mL DM
  • 78 µL 35% BSA (to 0.04%)
• DM+BSA+Optiprep 20% (total 1.8 mL: 0.3 mL per tube, x6 for 3 regions x 2 hemispheres)
  • 3.2 mL DM+BSA
  • 800 µL Optiprep

Procedure

• Perfusion & Dissection (work in pairs with 2 vibratomes: 1 person slicing, 1 person dissecting)

1. Perfuse the mouse with 10 - 15 mL cold choline perfusion solution.

• * Change the tubing on the peristaltic pump to avoid contaminating shared lab tubing with drugs added to the perfusion solution.

2. Dissect out the brain in cold choline solution.

3. Make a blocking cut for coronal sectioning. Keep the cut towards the posterior side of the cortex to get Hb slices (approx. over the middle/posterior half of VCtx).

4. Glue the brain to the vibratome cutting disk. 

5. Cut off a chunk from the ventral side of the right hemisphere.

6. Slice at 500 µm. Transfer slices to a small petri dish filled with choline dissection solution.

7. Transfer the slices into the dissection dish filled with choline dissection solution. Keep the dissection dish in a 15 cm dish filled with ice.

8. Dissect out mPFC/MCtx/Hb separately for each hemisphere and transfer each region into its corresponding tube using a microdissection knife into a tube with 3.3 mL DM (+ 1 µM TTX, + 10 µM CPP, + 5 µg/mL Actinomycin D, + 10 µM Triptolide, + 30 µg/mL Anisomycin). Keep the tubes on ice during the dissection.

• - Use either CPP or AP-V

9. Pool dissected regions across mice (4-6 mice for mPFC + MCtx, 6 mice for Hb).

• * Approx. 30-45 min per mouse pair.

• Dissociation

10. Add 3.3 mL of Papain 2x to the tube and transfer to 37°C. Gently rock for 60 min (90 min for Hb).

• * Seal the tube caps with parafilm to ensure that they do not leak.
• * Start the digest for mPFC and MCtx first, and then continue with the dissection for Hb (additional 4 mice, approx. 1 hour).

11. Transfer the tubes to ice.

12. Triturate at medium speed with a 10 mL pipette. Leave to settle.

13. Leaving 1 mL in the tube, filter remaining supernatant through a corner of a 40 µm filter (cell strainer).

14. Triturate the remaining 1 mL 10 times with a P1000. Leave to settle.

15. Leaving 200 µL in the tube, filter remaining supernatant through the corner of the same 40 µm filter.

16. Triturate the pellet 10 times with a P200 pipette.

17. Filter the suspension through the same corner of the 40 µm filter.

18. Add 1 mL of TI 1:10 to the filter to wash off any remaining cells.

• * Use the pipette to draw out leftover drops from the underside of the filter.

19. Add an additional 1 mL of TI 1:10 to the filter for a 2nd wash.

20. Spin down at 300 g for 5 min.

• Purification/Clean-Up

21. Resuspend in 1 mL of TI 1:10 with a P1000.

22. Add 2 mL of TI 1:10.

23. Overlay gently on top of 5 mL TI.

• * Gently wet the tip of the pipette, and then pipette the resuspended pellet in TI 1:10 slowly down the side. Gently tap and swirl to slightly mix at the liquid boundary.

24. Spin down at 70 g for 10 min at max accel. & deccel.

25. Keep the supernatant. Resuspend pellet in 1 mL DM+BSA.

26. Count cells on the haemocytometer. Check if there are at least 30K cells/mL

27. If the density is too low (<30K/mL), take the TI supernatant and spin down at 850 rpm/300 g for 3 minutes. Resuspend the pellet in 500 µL DM and combine the samples.

28. Add 9 mL DM+BSA.

29. Spin down at 300 g for 5 min.

30. Resuspend in 100 µL DM+BSA (alternatively, re-suspend in leftover volume after removing the supernatant and measure it).

31. Add 3 x 100 µL (or 3x the measured volume) of DM+BSA+20% optiprep. Swirl as solution is added.

32. Count cells and dilute further with DM+BSA+15% optiprep down to ~ 100K cells/mL if necessary.

33. Transfer to inDrops machine.