Jun 02, 2023
Manual isolation of nuclei from human brain using CellRaft device and single nucleus Whole Genome Amplification
- Ester Kalef-Ezra1,2,
- Diego Perez-Rodriguez1,
- Christos Proukakis1,2
- 1Department of Clinical and Movement Neurosciences, Queen Square Institute of Neurology, University College London, London, UK;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
- Ester Kalef-Ezra: e.kalef-ezra@ucl.ac.uk;
- Diego Perez-Rodriguez: d.rodriguez@ucl.ac.uk
- Christos Proukakis: c.proukakis@ucl.ac.uk
Protocol Citation: Ester Kalef-Ezra, Diego Perez-Rodriguez, Christos Proukakis 2023. Manual isolation of nuclei from human brain using CellRaft device and single nucleus Whole Genome Amplification. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxzjjov8j/v1
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: August 11, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 68508
Keywords: Single cell, Whole Genome Amplification, Cell Raft, Somatic mutation, ASAPCRN
Funders Acknowledgement:
This research was funded in part by Aligning Science Across Parkinson’s through the Michael J. Fox Foundation for Parkinson’s Research (MJFF).
Grant ID: 000430
Abstract
Protocol for manual nuclear isolation from human brain tissue using Cell Raft device for single cell Whole Genome Amplification.
Attachments
Main Protocol.docx
1.2MB
dMDA version 2.pdf
317KB
CytoSort Array.pdf
7.5MB
CellRaft.pdf
714KB
PTA.pdf
2.6MB
Guidelines
Critical note: Unless otherwise indicated, all the reagents must be kept at 4ºC and all the steps in the protocol must be performed at 4ºC. To preserve nuclear integrity, all the solutions are supplemented with complete cOmplete EDTA-free Protease Inhibitor Cocktail.
References:
- Mullen RJ, Buck CR, Smith AM. NeuN, a neuronal specific nuclear protein in vertebrates. Development. 1992;116(1):201-211. doi:10.1242/dev.116.1.201.
- Wolf HK, Buslei R, Schmidt-Kastner R, et al. NeuN: a useful neuronal marker for diagnostic histopathology. J Histochem Cytochem. 1996;44(10):1167-1171. doi:10.1177/44.10.8813082.
- Cannon JR, Greenamyre JT. NeuN is not a reliable marker of dopamine neurons in rat substantia nigra. Neurosci Lett. 2009;464(1):14-17.
- Reed PJ, Wang M, Erwin JA et al (2017) Single-Cell Whole Genome Amplification and Sequencing to Study Neuronal Mosaicism and Diversity. In: Frade J., Gage F. (eds) Genomic Mosaicism in Neurons and Other Cell Types. Neuromethods, vol 131. Humana Press, New York, NY.
Materials
Commercial Reagents:
| A | B | C | D | E | |
| Item | Supplier | Catalogue Number | Preparation prior use | Storage | |
| UltraPure DNase/RNase-Free Distilled Water | Thermo Fisher Scientific | 10977049 | Aliquot | RT | |
| PBS(Phosphate Buffered Saline) 10X Solution (pH 7.4) | Fisher Scientific | 15815418 | Make 1x with dH2O | Fridge | |
| 50x cOmplete Protease Inhibitor Cocktail EDTA-free | Roche via Sigma Aldrich | 4693159001 | Use 1 tablet in 1 ml dH2O | Freezer (20oC) | |
| Triton-X100 | Sigma Aldrich | T9287 | Prepare10% aliquot | RT | |
| ODGM(Optiprep Density Gradient Medium) | Sigma Aldrich | D1556 | Aliquot | Fridge | |
| Goat Serum | Sigma Aldrich | G9023 | Aliquot | Freezer (20oC) | |
| Cell-Tak™ Cell and Tissu Adhesive | Corning via Sigma Aldrich | DLW354242 | No | Fridge | |
| DNase I Solution (1 unit/μL), RNase-free | Thermo Fisher Scientific | 89836 | Dilute to DNase solution, 200 U/mL and Aliquot | Freezer (20oC) | |
| TE Buffer (low EDTA) | Thermo Fisher Scientific | 12090-015 | Aliquot | RT |
UltraPure™ DNase/RNase-Free Distilled WaterThermo FisherCatalog #10977049
PBS (Phosphate Buffered Saline) 10X Solution (pH 7.4)Fisher ScientificCatalog #15815418
cOmplete mini EDTA free protease inhibitor cocktailSigma AldrichCatalog #4693159001 ,
OptiPrep™ Density Gradient MediumSigma AldrichCatalog #D1556
Goat serumSigma AldrichCatalog #G9023
DNase I Solution (1 unit/µL), RNase-freeThermo FisherCatalog #89836
Low TE BufferInvitrogen - Thermo FisherCatalog #12090-015
Home-Made Reagents:
| A | B | C | |
| Item | Sterilization Method | Storage | |
| 1 M MgCl2 | Autoclave | RT | |
| 1 M Tris/HCl pH 8.8 | Autoclave | RT | |
| 1 M Sucrose | Filtration | Freezer (-20oC) | |
| 1 M KCl | Autoclave | RT | |
| 1 mg/ml DAPI (4′,6-diamidino-2-phenylindole) | No | Freezer (-20oC) | |
| 10% Presept | No | RT | |
| 0.1 M NaOH solution | Filtration | Fridge | |
| 2.5 mM NaOH in PBS | Filtration | Fridge | |
| 70% EtOH in dH2O | No | RT |
Antibodies for immunodetection
| A | B | C | D | E | |
| Antibody | Supplier | Catalogue Number | Produced in | Working Concentration | |
| Sox6 (dopaminergic neuronal nuclei) | Merck | HPA001923 | Rabbit | 0.5 μg/ml | |
| Olig2 (oligodendrocyte nuclei) | Abcam | Ab109186 | Rabbit | 1 μg/ml | |
| aSyn (alpha-synuclein) | Santa Cruz Biotechnology | sc-12767 | Mouse | 1 μg/ml | |
| NeuN clone A60 (cortical neuronal nuclei)* | Millipore | MAB377 | Mouse | 10 μg/ml | |
| Secondary anti-mouse IgG conjugated with Alexa (568) | Life Technologies | A21235 | Goat | 2 μg/ml | |
| Secondary anti-rabbit IgG conjugated with Alexa (488) | Life Technologies | A11008 | Goat | 2 μg/ml |
Note
*Note: According to MAB377X description, this antibody works for most neuronal cell types throughout the adult nervous system. However, some neurons fail to be recognized by NeuN at all ages: INL retinal cells, Cajal-Retzius cells, Purkinje cells, inferior olivary and dentate nucleus neurons, and sympathetic ganglion cells and dopaminergic neurons (Mullen et al., 1992; Wolf et al., 1996., Cannon et al., 2009)
aSyn (alpha-synuclein)Santa Cruz BiotechnologyCatalog #sc-12767
Anti-NeuN Antibody, clone A60Merck MilliporeCatalog #MAB377
Specialized consumables:
- CytoSort 200, single, 5-pack - Cell Microsystems - 20200301CS.
General consumables:
- Low binding filtered tips (sterile).
- Low-binding 1.5ml and 0.2 ml tubes (sterile).
- Screwed lid transparent 1.5-2 ml tubes (sterile).
- 1ml syringe without needle (sterile).
- PES Syringe filter, 0.2 µm (sterile).
- Gloves.
- Strong glue e.g., super glue.
- Adhesive tape.
Equipment:
- Tissue culture hood for human sample handling.
- PCR cabinet (Here we used Air Science, Lydiate, UK).
- Dounce tissue grinder set 2 mL (Kimble via Sigma Aldrich - D8938).
- Refrigerated centrifuge for 1.5ml tubes that can achieve at least 13000 x g (here used Sigma Aldrich 1 - 14K Refrigerated Micro Centrifuge).
- Orbital Shaker (Here we used Grant instruments Orbital Shaker PSU-10i).
- Haemocytometer.
- Inverted microscope (Here we used Nikon Eclipse TE300 inverted microscope coupled to a CCD camera - KERN optics).
- CellRaft accessory device (Cell Microsystems, Durham, US), with single reservoir CytoSort array (10,000 rafts).
- Stereoscope.
- General lab pipettes.
- Pair of forceps and scissors.
Safety warnings
Please follow the Safety Data Sheets (SDS) for all reagents for safe handling and safety hazards.
This procedure was adapted from:
• Wierman MB, Burbulis IE, Chronister WD, Bekiranov S, MJ MC (2017) Single cell CNV detection in neuronal nuclei. In: Springer (ed) Genomic Mosaicism in Neurons and Cell Types (editors: Frade JM, Gage FH). New York, USA: Humana Press, Springer Nature; ISBN 978-1-4939-7279-1.
• Perez-Rodriguez D, Kalyva M, Leija-Salazar M, et al. Investigation of somatic CNVs in brains of synucleinopathy cases using targeted SNCA analysis and single cell sequencing. Acta Neuropathol Commun. 2019;7(1):219. Published 2019 Dec 23.
• Perez-Rodriguez D, Kalyva M, Santucci C, Proukakis C (2022) Somatic CNV Detection by Single-Cell Whole-Genome Sequencing in Postmortem Human Brain. In: Methods in Molecular Biology: Alzheimer’s disease. Vol. 2561, Jerold Chun (Ed).
Before start
Section 1: Nuclear extraction from human brain
Before starting:
1. Clean Human Tissue handling hood with 0.2 M NaOH, 10% Presept, 70% EtOH, and dH2O.
2. Clean Dounce tissue grinders (prior to use, between samples, and at the end of the experiment) with 0.2 M NaOH, 10% Presept, 70% EtOH, dH2O, and let them dry.
3. UV sterilize and clean PCR hood, centrifuge, pipettes, etc. with 10% Presept, 70% EtOH, and dH2O.
4. Precool centrifuge @4ºC.
5. On the day of the experiment prepare the following Buffers and keep themon ice:
- NIM (Nuclear Isolation Media): 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl pH 8.8, 250 mM sucrose, 1 mM dithiothreitol (DTT), 1x cOmplete EDTA-free Protease Inhibitor Cocktail.
- ODN (Optiprep Diluent for Nuclei): 150 mM KCl, 30 mM MgCl2, 60 mM Tris-HCl pH 8.8, 250 mM sucrose, 1x cOmplete EDTA-free Protease Inhibitor Cocktail.
- 25% iodixanol solution (NIM:ODGN:ODN 6:5:1).
- 29% iodixanol solution (ODGN: ODN 29:31).
- NSB (Nuclei Storage Buffer) - if needed: 5 mM MgCl2, 50 mM Tris-HCl (pH 8.8), 166 mM sucrose and 1 mM dithiothreitol (DTT), 1x cOmplete EDTA-free Protease Inhibitor Cocktail.
C. Proceed to Immunodetection of inclusions and/or cell type marker staining
Before starting:
- Prepare PBS: PBS 1x. Keep on ice until use.
- Prepare PBS/PIC: PBS 1x supplemented with 1x cOmplete EDTA-free Protease Inhibitor Cocktail. Keep on ice until use.
- Prepare Blocking Buffer: PBS+PIC supplemented with 10% goat serum. Keep on ice until use.
Section 3. View CytoSort Arrays under the microscope, capture & transfer rafts with single nuclei in tubes for single cell Whole Genome Amplification
Before starting:
- Defreeze an aliquot of diluted DNase I Solution and keep itat RT.
- Prepare aliquots of 50 µl of 70% EtOH and 50 µl of dH2O in 1.5ml tubes in a PCR hood and keep at RT until use. You will need at least 1 aliquot of 70% EtOH and dH2O for each sample you want to amplify. As an example, if you aim to amplify the genome of 10 single nuclei, prepare 15 aliquots of 50 µl of 70% EtOH and 50 µl of dH2O.
- Prepare 0.2ml tubes with buffer depending on the downstream single cell Whole Genome Amplification (scWGA) method: (estimate double the number of tubes than the single nuclei to be collected):
a. Multiple Displacement Amplification (MDA) in droplets (dMDA, Samplix): 2.8µl Lysis Bf (200 mM KOH, 5 mM EDTA pH 8 and 40 mM 1.4 DTT) diluted in 2 µl dH2O
b. PicoPlex (Takara): 5µl TE/Cell Extraction Buffer (depending on the version of the PicoPlex protocol used)
c. Primary Template-directed Amplification/Resolve DNA (PTA, BioSkryB): 3µl Cell Buffer (BioSkryB)
- Mount the CellRaft® Release Device onto a 4× or 10× objective of an inverted microscope and prepare the CytoSort Array for observation under the microscope. For details on this process please see the attached protocols (Attach CytoSort Array and CellRaft protocols):
1. Cell Microsystems: CellRaft System for Inverted Microscopes (user manual)
2. Cell Microsystems: CytoSort Array (user manual).
Figure 1. A CytoSort Array is attached to the microscope to allow observation of the rafts and manual picking using a magnetic wand.
Nuclear extraction from human brain:
Nuclear extraction from human brain:
28m
28m
Transfer 20 mg -50 mg frozen brain tissue to microcentrifuge tube containing 500 µL of NIM. Transfer the tissue in a tube with the lid on using a pair of forceps.
Optional step: Mincing the tissue in small fragments over a glass plate On ice using a scalpel could significantly decrease the time needed for homogenization and facilitate the trituration with the pipette tips.
Gently triturate the tissue with a cut pipette tip (1 mL tip). Repeat several times with pipette tips cut less (estimate 3-4 different cut sizes).
Note
Tissue will be perfectly dissociated when able to pass through an uncut pipette tip.
Measure the total volume using 1 mL pipette.
Add Triton X-100 solution to the sample to achieve final concentration 0.1% (stock solution 10%).
Homogenize using 2 mL Dounce tissue grinders:
a. Grinder A (loose, large clearance pestles): 2-3 strokes.
b. Grinder B (tight, small clearance pestles): 8-12 strokes.
Note
Note: The sample solution should appear homogeneous at this point, but the use of the Dounce grinders allow nuclei to remain intact.
Transfer the homogenate to a fresh 1.5 mL microcentrifuge tube.
Centrifuge at 1000 x g , 00:08:00 , at 4 °C - Discard Supernatant.
8m
Resuspend the pellet gently in 700 µL 25% Iodixanol Solution.
Layer the sample (avoiding mixing the phases) onto 700 µL 29% iodixanol solution. Use 1 mL syringe without needle to slowly load and suspend the sample on top. As a result, 2 clearly defined phases should appear as follows:
Figure 2: Nuclear isolation using iodixanol gradient. A. Gradient generation solution is generated from 25% iodixanol gradient containing the cell lysate on the top and 29% iodixanol gradient solution on the bottom. B. After centrifugation, the nuclear fraction is near the bottom of the tube.
Spin at 10300 x g , 00:20:00 , at 4 °C - remove the supernatant leaving 50 µL in the bottom of the ultracentrifuge tube (contain the nuclear fraction).
20m
At this point, follow any of the below routes:
Store nuclear suspensions @4ºC30 steps
Section 1, step 12, Option A
Note
Note: Although we routinely use the nuclear suspensions on the day of preparation, others have reported those to be stable at at 4 °C for several weeks when stored in nuclei storage buffer (Reed et al., 2017).
Estimate nuclei number under the microscope:
Estimate nuclei number under the microscope:
Use a haemocytometer to estimate the number of nuclei in the final suspension. In a good nuclear fraction, most of the nuclei should be seen as single, with few clumps and/or low amount of cellular/tissue debris.
Manual isolation of single nuclei using a device fixed to an inverted microscope:
Manual isolation of single nuclei using a device fixed to an inverted microscope:
Note
Critical note: If possible, do sub-sections 1 and 2 in a PCR cabinet to minimize contamination.
Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
Visually inspect the CytoSort array prior opening. The pre-sterilized arrays are expected to have a red sticker on packaging. Remove the array from the sealed pouch.
Figure 3. Examples of CytoSort arrays prior to opening (A) without, and (B) with crystallization.
Optional step: It may be possible to observe crystallization of excess glucose that was used to seal the CytoSort array from exposed air (Figure 2B). In that case:
Wash the array with 2 mL (pre-warmed at 37 °C ) PBS 1x – incubate for 00:03:00 .
3m
Repeat above step 3 times in total:
Wash the array with 2 mL (pre-warmed at 37 °C ) PBS 1x – incubate for 00:03:00 (1/3).
3m
Wash the array with 2 mL (pre-warmed at 37 °C ) PBS 1x – incubate for 00:03:00 (2/3).
3m
Wash the array with 2 mL (pre-warmed at 37 °C ) PBS 1x – incubate for 00:03:00 (3/3).
3m
Cool the array at Room temperature .
Continue to the next steps.
Wash with 2 mL dH2O – incubate for 00:03:00 at Room temperature – Remove water.
3m
Repeat the washing with 2 mL dH2O – incubate for 00:03:00 at Room temperature - Remove water.
3m
Apply 1 mL of 15 ug/mL Cell-Tak in PBS with 2.5 millimolar (mM) NaOH – incubate for 00:20:00 @Room temperature – Remove Cell-Talk.
20m
Add 2 mL sterile PBS 1x - Incubate for 00:03:00 at Room temperature – Remove PBS.
3m
Repeat step 21 (3 times in total):
Add 2 mL sterile PBS 1x - Incubate for 00:03:00 at Room temperature – Remove PBS (1/3).
3m
Add 2 mL sterile PBS 1x - Incubate for 00:03:00 at Room temperature – Remove PBS (2/3).
3m
Add 2 mL sterile PBS 1x - Incubate for 00:03:00 at Room temperature – Remove PBS (3/3).
3m
Manual isolation of single nuclei using a device fixed to an inverted microscope-Seeding nuclei to the CytoSort array:
Manual isolation of single nuclei using a device fixed to an inverted microscope-Seeding nuclei to the CytoSort array:
Optional step: Insert the CellRaft seeding insert to increase the overall number of single cells available for isolation.
Dilute approximately 5000 nuclei in 2 mL of PBS, add pipette them dropwise in the centre of the CytoSort Array.
Note
Critical note: Nuclei should be counted in a haemocytometer prior to seeding. The number of nuclei seeded on the array is a key factor, as too many will lead to few CellRafts with single nuclei, while too little will lead to most of the CellRafts being empty, according to a Poisson distribution. Thus, seeding the array with a nuclei: wells ratio 1:2 or 1:3 increases the proportion of wells capturing a single nucleus versus wells capturing none or >2 nuclei.
Move the CytoSort Array carefully to ensure the nuclei settle evenly within the array (like seeding cells for cell culture).
Allow it to settle for 02:00:00 - O/N in the fridge.
Note
Note 1: Use parafilm, paper towel and foil to properly seal the array to prevent desiccation
Note 2: From our experience, nuclei are stable on the array for up to 336:00:00 when stored properly sealed in the fridge.
2h
Isolation of rafts containing the nuclei of interest:
Isolation of rafts containing the nuclei of interest:
20m
20m
View CytoSort Arrays under the 10x magnification objective with appropriate fluorescence filters, to select rafts (microwells) containing a single nucleus with the desired characteristics and take photos (if needed Fig 4).
Figure 4. Manual picking of rafts containing the nuclei of interest. A CellRaft with a single nucleus stained by DAPI.
Individually release the arrays of interest (Figure 5).
Figure 5. Example of released and removed CellRafts.
Place the magnetic wand in the tubes containing DNase-I diluted solution.
Note
Critical note: DNAse-I solution is used to reduce cross-contamination between samples. This may require optimisation in each lab.
Wash the wand with 70% EtOH and then with dH2O.
Capture, release and remove the released raft to the already prepared 0.2 mL tubes with the buffer needed for scWGA using the magnetic want. Then, centrifuge the tubes briefly.
When performing CellRaft isolation, it is highly recommended to ensure the rafts are properly released at the bottom of the tube and not in the tube wall, as they would desiccate. To do so, visually inspect every tube after collection under a stereoscopic microscope; rafts will appear as small orange squares.
Note
Critical note: As controls, use at least one of each of the following per experiment:
- Negative control 1: a tube with no raft, in which the magnetic wand has been dipped after it was dipped into the array covering fluid.
- Negative control 2: a raft with no nucleus.
- Positive control: 15 pg of genomic DNA.
Figure 6. Wand washing, raft picking and release in 0.2 ml tubes.
Freeze the samples at -80 °C or proceed directly to scWGA.
For scWGA we have used the following methodologies:
- PicoPlex (R300670, R300672 and R300722) according to Takara’s protocols.
- dMDA with heat denaturation and Buffer Generation (dMDA protocol attached).
- PTA (PTA protocol attached) but for step 7: incubate for 00:20:00 at Room temperature .
Note
Note: So far, we have successfully used the PicoPlex method with and without nuclei immunostaining. However, we have not tested the dMDA and PTA methods in combination with immunostaining, but we have just stained the nuclei with DAPI staining.
20m