Zebrafish retinal dissociation, cell sorting, and methanol fixation

Dissociation

• For all tubes containing cells, use LoBind tubes (Eppendorf 022-43-102-1)
• Add 5 ml PBS (pH7.4) to papain vial (Worthington Biochemical, LK003178), place at 37°C until retinas are dissected 

• Dissect retinas, placing up to 10 retinas in 0.8 ml Leibowitz media on ice (Thermo Fischer Scientific, 21083027)
• Add 89 μl of 10 mg/ml hyaluronidase (Sigma-Aldrich H3884-50MG) to each tube, place on rocker for 15 minutes at room temperature
• Allow retinas to settle to bottom, remove supernatant with fire-polished pipette (avoid pipetting any tissue, a small volume of supernatant remaining is acceptable)
• Add 50 μl 10,000 U/ml DNaseI (Roche 04536282001) to papain vial (if not using full 5ml of papain, make 1ml aliquots and add 10ul DNaseI to each aliquot that will be used immediately, unused papain solution can be stored at -20°C for future use within 2 weeks, don't add DNaseI to papain solution that won't be used)
• Add 1 ml papain/DNase solution to tube containing the retinas. Place horizontally in incubator shaker at 28.5°C for 30 min at 450 rpm 
• Triturate gently 3 times using fire-polished pipette
• Pellet 1500 xg at 4°C for 10 min (dislodge and repeat if compact pellet doesn't form)
• Remove supernatant and add 500 μl of inhibitor solution to tubes:
  4.2 μl 1M MgCl₂
  10 μl 10mg/ml leupeptin (Sigma-Aldrich L2023-10MG)
  10 μl DNaseI (10,000U/ml)
  975.8 μl PBS (pH7.4)
• Tap gently to dislodge pellet, triturate gently 3 times with fire-polished pipette if pellet isn't dislodging. DNA may make sample sticky, this is normal and will be removed during next incubation
• Combine tubes if they are the same sample
• Leave at room temp for 10 mins (helps break down extracellular DNA), pass through 70 μm filter (Miltenyi Biotec 130-098-462) (proceed to methanol fixation if for single cell)
• For cell sorting, take 50 μl of sample and add 450 μl PBS (unstained sample, required for gating)
• Add 2 μl 1mg/ml Propidium Iodide (Life Technologies, P1304MP; labels dead cells) to sample (PI stained sample)
• Prepare same for non-fluorescent controls if sorting cells based on fluorescence (required for gating)
• Place all samples on ice
• Sort into Trizol-LS (Life Technologies 10296-010; volume depends on volume of cells collected) for RNA isolation and freeze on dry ice, or FBS (2:1 FBS:PBS) for collecting whole cells, keep on ice

Methanol Fixation for scRNA-Seq

• Centrifuge cells 800 xg at 4°C for  3 min
• Remove supernatant, add 1 ml 4°C D-PBS (Thermo-Fischer Scientific, 14190144), resuspend cells by pipetting gently
• Centrifuge cells 800 xg at 4°C for 5 min
• Remove supernatant, add 1 μl 4°C D-PBS, resuspend cells by pipetting
• Centrifuge cells 800 xg at 4°C for 5 min
• Remove supernatant, add 100 ml 4°C D-PBS, resuspend cells by pipetting
• Add 900 μl -20°C 100% methanol (Sigma-Aldrich 34860-100ML-R) dropwise, while vortexing slowly (prevents cells clumping), transfer to cryovial (VWR international 5000-0012)
• Leave on ice for 15 min, then place in -80°C (sample will remain as liquid, and can be moved or shipped on dry ice)

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