Oct 30, 2023

Public workspaceVillage Nuclei Isolation With Optiprep

DOI
dx.doi.org/10.17504/protocols.io.36wgq3bmxlk5/v1
  • 1Harvard Medical School
liv_spina
Open access
DOI: dx.doi.org/10.17504/protocols.io.36wgq3bmxlk5/v1
Protocol Citation: liv_spina, Tara McDonald, Nora Reed, Alyssa Lutservitz 2023. Village Nuclei Isolation With Optiprep. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3bmxlk5/v1
Manuscript citation:
Wells et al., PMID 36796362
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 30, 2023
Last Modified: October 30, 2023
Protocol Integer ID: 90142
Keywords: fresh-frozen nuclei, nuclei extraction, snRNAseq, RNAseq, human tissue, brain tissue, multiple donors
Abstract
Isolation of nuclei from fresh-frozen brain tissue from sets of multiple (typically 2-20) human donors for analysis as a “cell village” (Wells et al., PMID 36796362) in which nuclei from all donors are analyzed together. Adapted from dx.doi.org/10.17504/protocols.io.bs99nh96, Luciano G Martelotto, with modifications to optimize for human brain tissue and allow the “cell village” approach.
Materials
Supplies
  • Scalpels
  • Glass slides
  • Dounce
  • 20 µm vacuum filter
  • Eppendorf tubes (1.5 mL and 5 mL)
  • Eppendorf or Ranin pipette tips
  • Dry ice
  • Metal plate
  • OCT (Optimal cutting temperature compound)
  • RNAse free water
  • Cell counting supplies (LUNA-FL)

Other Reagents:
  • PBS
  • BSA
  • RNAse inhibitor (i)
  • Nuclei EZ lysis buffer (NUC201-1KT)
  • OptiPrep Density Gradient Medium (60% Iodixanol - G60) (ab286850)

Stock solutions:
  • 500 mM tricine: 8.96 g of tricine in 100 mL water
  • 1 M KCl: 7.45 g of KCl in 100 mL water
  • 1 M MgCl2x6H2O: 20.3 g of MgCl2x6H2O in 100 mL water
Before start
  • Turn on and chill centrifuge to 4℃
  • Prepare all the reagents needed on ice
  • Clean glass slides with ethanol
  • Gather the dounce, pestles, and scalpels on ice
Solutions to make fresh before starting experiment:
  • Nuclei EZ lysis buffer -- Pour 12 mL of buffer into the dounce and reserve 1 mL for wetting down the sides of the dounce
  • PBSA + i (1% BSA and 1 U/µL RNase inhibitor in 1X PBS) -- For powder BSA, add 0.01 g of BSA per 1 mL of 1X PBS
  • G30: (Mix the G60 and GD together thoroughly before adding the GH)
Before Starting
Before Starting
Gather Supplies
  • Scalpels
  • Glass slides
  • 14 mL Dounce
  • 20 µm vacuum filter
  • Eppendorf tubes (1.5 mL and 5 mL)
  • Eppendorf or Rainin pipette tips
  • Dry ice
  • Metal plate
  • OCT (Optimal cutting temperature compound)
  • RNAse free water
  • Cell counting supplies (LUNA-FL)
Other Reagents:
  • PBS
  • BSA
  • RNAse inhibitor (i)
  • Nuclei EZ lysis buffer (NUC201-1KT)
  • OptiPrep Density Gradient Medium (60% Iodixanol - G60) (ab286850)
Stock solutions:
  • 500 mM tricine: 8.96 g of tricine in 100 mL water
  • 1 M KCl: 7.45 g of KCl in 100 mL water
  • 1 M MgCl2x6H2O: 20.3 g of MgCl2x6H2O in 100 mL water
Optiprep Buffers (can be made in advance and kept on hand)
ABCD
GDGH
ComponentVolumeComponentVolume
500 mM Tricine24 mLSucrose8.5 g
1M KCl15 mLWater50 mL
1M MgCl2 x 6H2O3 mL500 mM Tricine4 mL
Water50 mLKCl2.5 mL
1M KOHAdjust to pH 7.81M MgCl2x6H2O0.5 mL
Bring volume up to 100 mL with water (keep at room temp)1M KOHAdjust to pH 7.8
Bring volume up to 100 mL with water (keep at 4°C)

  • Turn on and chill centrifuge to 4℃
  • Prepare all the reagents needed on ice
  • Clean glass slides with ethanol
  • Gather the dounce, pestles, and scalpels on ice
Solutions to make fresh before starting experiment
Nuclei EZ lysis buffer
  • Pour 12 mL of buffer into the dounce and reserve 1 mL for wetting down the sides of the dounce
PBSA + i (1% BSA and 1 U/µL RNase inhibitor in 1X PBS)
  • For powder BSA, add 0.01 g of BSA per 1 mL of 1X PBS
G30: (Mix the G60 and GD together thoroughly before adding the GH)

ABCDEF
G30PBSA + iNuclei EZ Lysis Buffer
ComponentVolumeComponentVolumeComponentVolume
G60 (OptiPrep)6.0 mLPBS (pH 7.4) (1X)1 mL of 10X PBSEZ Lysis Buffer12,975 µL
GD1.2 mLBSA (100X) 1 mL of 10% BSARNAse Inhibitor325 µL
GH4.8 mLRNase Inhibitor (200X) 50 µL
RNase free water7,950 µL

Tissue Homogenization
Tissue Homogenization
1h 30m
On a glass slide on dry ice, shave off pieces of tissue with a scalpel until you have 50±3 mg of tissue
For managing tissue, use OCT to adhere tissue to glass slide
Place tissue pieces on a boat on dry ice. Hold the tissue on dry ice until all tissue has been cut
Transfer all tissue to dounce filled with Nuclei EZ Lysis Buffer
Use 1mL of the reserved Nuclei EZ Lysis Buffer to wet down the side of the dounce if tissue is stuck on the side of the dounce
Gently dounce tissue on ice 20 times with pestle “A” then 20 times with pestle “B” until the tissue is homogenized
Incubate on ice for 10 minutes
Nuclei Isolation
Nuclei Isolation
1h 30m
Wet a 20 µm vacuum filter with 1 mL of PBSA + i
1h
Vacuum filter the homogenized tissue
If the filter clogs, remove any unfiltered volume with a pipette, replace the filter, and continue filtering
Transfer the homogenized tissue to (3) 5 mL tubes
Centrifuge the samples at 4℃ for 5 min (500 x g)
Remove and discard the supernatant (be careful not to disturb cell pellets) and resuspend each pellet in 900 µL G30
Alternatively, split pellet into (9) 1.5 mL tubes (total), resuspend each pellet in 300 µL of G30, then underlay each sample with 1 mL of G30
Gently underlay each 5mL tube with 3 mL G30

Place the pipette tip filled with G30 at the bottom of the 5mL tube. Slowly dispense G30 underneath the cell suspension. A clear separation will form between homogenate and G30 (cloudy on top, clear on the bottom). Do not disturb the separation between the layers
Centrifuge the samples at 4℃ for 10 min (4,255 x g)
For capturing smaller cell types, spin at 8,000 x g for 20 minutes
Remove and discard the supernatant
Remove the top (very viscous) layer from the tube first- this layer contains fats and must be removed before removing the rest of the supernatant
Nuclei Wash, Resuspension, and Quantification
Nuclei Wash, Resuspension, and Quantification
20m
Resuspend the pellets in 1 mL PBSA + i
Centrifuge the samples at 4℃ for 5 min (500 x g) (single wash)
Remove the supernatant and resuspend the pellets in 200 - 1,000 µL of PBSA + i (depending on the size of the pellet)
Count nuclei using LUNA-FL counter
Preserving Leftover Nuclei
Preserving Leftover Nuclei
15m
Optional if you have excess nuclei, or if you need to pause before continuing.
Centrifuge the nuclei at 4℃ for 5 min (500 x g)
Remove the supernatant and resuspend the nuclei in CryoStor (CryoStor® CS10), mix by pipetting
≤ 1k nuclei / µL of CryoStor
Transfer the nuclei to cryotubes and store them in a Mr Frosty in a -80℃ freezer until frozen (1 day)
For long term storage, transfer cryotubes to regular storage in a -80℃ freezer
Thawing Cryo-Stored Nuclei
Thawing Cryo-Stored Nuclei
30m
Thaw the CryoStored nuclei
Centrifuge the nuclei at 4°C for 10 min (1200 x g)
Remove the supernatant and resuspend in 1mL PBSA + i
Centrifuge the nuclei at 4°C for 5 min (500 x g) (single wash)
Remove the supernatant and resuspend the pelleted nuclei in PBSA + i
Count the nuclei before proceeding to next steps
Protocol references
Wells et al., PMID 36796362
Adapted from dx.doi.org/10.17504/protocols.io.bs99nh96, Luciano G Martelotto