Mar 29, 2023
A versatile nuclei extraction protocol for single cell multiome ATAC and gene expression in non model species
- Rose Ruiz Daniels1,
- Richard S Taylor1,
- Ioannis Konstantinidis2,
- Sarah Salisbury1,
- Diego Perojil Morata1,
- Jorge Manuel de Oliveira Fernandes2,
- Emily Clark1,
- Dan Macqueen1,
- Diego Robledo1
- 1The Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Edinburgh EH25 9RG, UK;
- 2Nørd University, Bodø, Hovedbygning 2060, Norway
Protocol Citation: Rose Ruiz Daniels, Richard S Taylor, Ioannis Konstantinidis, Sarah Salisbury, Diego Perojil Morata, Jorge Manuel de Oliveira Fernandes, Emily Clark, Dan Macqueen, Diego Robledo 2023. A versatile nuclei extraction protocol for single cell multiome ATAC and gene expression in non model species . protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l69wnrlqe/v1
Manuscript citation:
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: March 28, 2023
Last Modified: March 29, 2023
Protocol Integer ID: 79548
Keywords: snRNA-seq , aquaculture, non-model species, nuclei
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Abstract
Here we present a modified version of : dx.doi.org/10.17504/protocols.io.261genwm7g47/v2 that was used to successfully extract nuclei from an array of different tissue types for single cell sequencing and modify it with the purpose of extracting nuclei for single cell multiome ATAC and gene expression on the 10x chromium. The modifications for this protocol include: different concentration of RNase inhibitors, different quantities for nuclear isolation buffer, removal of unnecessary steps as well as QC specific for multiome analysis.
If you are looking to use this protocol for bulk ATAC-seq use of protease inhibitor cocktail PIC is recommended instead of RNase inhibitor on the snRNA-seq version of this protocol (dx.doi.org/10.17504/protocols.io.261genwm7g47/v2) please get in touch with the authors if you are unsure on how to do this.
Guidelines
Before using this prep for library preparation do a trial run.
It is recommended to conduct a trial, especially on a new tissue type, to adjust various parameters without introducing RNase. This allows for the adjustment of parameters such as mincing times, filter size, and dilution into the final buffer, which ultimately leads to the production of good quality nuclei.
For multiome snRNA-seq and single cell ATAC-seq, it is advisable to perform a trial run where the quality of the nuclei is assessed by examining their integrity and the quality of their nuclear membrane (as shown in the picture in step 2.1).
Materials
MATERIAL
Noyes Spring Scissors - Tungsten CarbideFine Science ToolsCatalog #15514-12
Tungsten Carbide Straight 11.5 cm Fine Scissors Fine Science ToolsCatalog #14558-11
40 µm Falcon™ Cell StrainersFisher ScientificCatalog #08-771-2
Corning™ Falcon™ Test Tube with 35µm Cell Strainer Snap CapCorningCatalog #352235
pluriStrainer Mini 20 µm (Cell Strainer)pluriSelectCatalog #43-10020-50
X500 Eppendorf DNA LoBind Tubes, 1.5ml, PCR clean
Cryotube
6-well tissue culture plate (Stem Cell Technologies)
Falcon tubes 15 ml (Corning)
INCYTO C-Chip™ Disposable HemacytometersVWR InternationalCatalog #82030-468
SAMPLING AND STORAGE FOR NUCLEAR ISOLATION
Animals must be appropriately euthanized and immediately processed. Approximately ~60 mg of salmonid tissue is placed in one clearly labelled cryotube and immediately flash frozen in liquid nitrogen. This step is critical. The tissue must be preserved as fast as possible for optimal results. In the absence of liquid nitrogen, samples can be frozen in dry ice. Samples can be stored at -80 °C for up to a year prior to use. Older samples might still yield viable nuclei but this would need to be tested.
REAGENTS
All reagents should be chilled on ice prior to use.
2X stock of salt-Tris solution makes 10 mL :
Stocks:
NaCl: NaCl (5 M) RNase-freeThermo Fisher ScientificCatalog #AM9759
Tris-HCl pH 7.5: UltraPure™ 1 M Tris-HCI Buffer, pH 7.5Thermo FisherCatalog #15567027
CaCl2: Calcium chloride 1 M in aqueous solutionVWR InternationalCatalog #97062-820
MgCl2: Magnesium chloride solution for molecular biology (1.00 M)Merck MilliporeSigma (Sigma-Aldrich)Catalog #M1028
Nuclease-free water: Water for biotechnology nuclease-free sterileVWR InternationalCatalog #97062-794
| A | B | C | |
| Stock solution (see above) | Volume | Final concentration | |
| NaCl | 292 ul | 146 mM | |
| Tris-HCL10 | 100 ul | 10 mM | |
| CaCl2 | 10 ul | 1 mM | |
| MgCl2 | 210 ul | 21 mM | |
| Nuclease-free water | 9388 ml |
The following buffers contain RNAase inhibitor Protector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335399001
- It is important to use the correct RNAse inhibitor as it can negatively affect library prep, check with the sequencing platform before using another type of RNAse.
- Do not add RNAse until right before nuclear extraction.
- RNAse inhibitor does not need to be used to test nuclear extractions, but it should added for sequencing runs.
1X ST buffer solution (ST) - 10 mL :
Dilute 2x ST in ultrapure nuclease-free water (1:1)
| A | B | C | |
| Stock Solution | Volume | Final concentration | |
| 2X ST | 3 ml | ||
| Ultrapure nuclease free water | 3 ml | ||
| RNAse inhibitor | 250 µl (240 U) | 200 Uml |
Make fresh and chill prior to use, add RNAnase inhibitor right before nuclear isolation. RNAase inhibitor amount can up upped if it’s an RNAse Rich tissue, up to 500 U per ml instead, tissue spends very little time in this buffer and is chilled at all time, which is why the amount of RNAase inhibitor can be lower.
Working solution (TST) – 4 mL :
1% Tween-20: Tween-20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P-7949
2% BSA: Bovine Serum Albumin (20 mg/mL) Molecular Biology GradeNew England BiolabsCatalog #B9000S
| A | B | C | |
| Stock solution | Volume | Final concentration | |
| 2X ST buffer | 2 ml | ||
| 1% Tween-20 | 120 µl | ||
| 2% BSA | 20 µl | ||
| Nuclease-free water | 1810 µl | ||
| RNAse inhibitor | 50 µl | 1000 Uml |
Make fresh and chill prior to use, add RNAase inhibitor right before nuclear isolation .Dilute the Tween from 10% in stock solution with nfH2O before making the buffer. RNAnase inhibitor amount can be upped if it’s an RNAase rich tissue up to 1000 U per ml instead, the nuclear isolation will happen in this buffer so its more critical in here.
Final dilution buffer will be stated on the protocol that the authors chose to use going forward, This protocol was developed using 10x multiome assay. RNAse inhibitor should be of concentration 1000 U/Ml, the RNAse inhibitor used should be protector RNase inhibitor (sigma-Aldrich)
Protocol materials
Corning™ Falcon™ Test Tube with 35µm Cell Strainer Snap CapCorningCatalog #352235
Materials
Water for biotechnology nuclease-free sterileVWR InternationalCatalog #97062-794
Materials
Noyes Spring Scissors - Tungsten CarbideFine Science ToolsCatalog #15514-12
Materials, Step 2
Magnesium chloride solution for molecular biology (1.00 M)Merck MilliporeSigma (Sigma-Aldrich)Catalog #M1028
Materials
Bovine Serum Albumin (20 mg/mL) Molecular Biology GradeNew England BiolabsCatalog #B9000S
Materials
Falcon™ Cell StrainersFisher ScientificCatalog #08-771-2
Materials
UltraPure™ 1 M Tris-HCI Buffer, pH 7.5Thermo FisherCatalog #15567027
Materials
Protector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335399001
Materials
Calcium chloride 1 M in aqueous solutionVWR InternationalCatalog #97062-820
Materials
Tween-20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P-7949
Materials
Tungsten Carbide Straight 11.5 cm Fine Scissors Fine Science ToolsCatalog #14558-11
Materials
INCYTO C-Chip™ Disposable HemacytometersVWR InternationalCatalog #82030-468
Materials
pluriStrainer Mini 20 µm (Cell Strainer)pluriSelectCatalog #43-10020-50
Materials
NaCl (5 M) RNase-freeThermo Fisher ScientificCatalog #AM9759
Materials
Before start
Sampling and storage for nuclear isolation.
Animals must be appropriately euthanized and immediately processed. Approximately ~60 mg of tissue is placed in one clearly labelled cryotube and immediately flash frozen in liquid nitrogen. This step is critical. The tissue must be preserved as fast as possible for optimal results. In the absence of liquid nitrogen, samples can be frozen in dry ice. Samples can be stored at -80 °C for up to a year prior to use. Older samples might still yield viable nuclei but this would need to be tested.
All reagents should be chilled on ice prior to use.
Samples should be kept frozen on dry ice until immediately before nuclei isolation, and all sample-handling steps should be performed on ice.
The centrifuge should be pre chilled at 4 °C .
All reagents are given for 2 nuclear isolations.
Amounts of buffer especially those that contain RNase should be adjusted appropriately for each experiment prepared prior and RNase added immediately before use.
Nucleus isolation workflow for ST-based buffers
Nucleus isolation workflow for ST-based buffers
30m
30m
Note
Samples should be kept frozen on dry ice until immediately before nuclei isolation, and all sample-handling steps should be performed On ice . The centrifuge should be pre-chilled at 4 °C .
On ice , place a piece of frozen tissue into one well of a 6-well tissue culture plate with 1 mL TST.
Note
If the sample is stuck to the cryotube, remove using tweezers, preferably while still in dry ice, and place immediately into the culture plate with TST. If the sample needs processing for examples cutting this is best done on dry ice. This is avoided by processing the sample prior to flash freezing.
On ice , mince tissue initially using Tungsten Carbide scissors for 00:00:30 and then with Noyes Spring Scissors Noyes Spring Scissors - Tungsten CarbideFine Science ToolsCatalog #15514-12 for a total of 00:10:00 .
Note
This step is only necessary for fin, skin or similar hard tissues, for softer tissues just use spring scissors for 00:10:00 .
10m
00:05:00 into the mincing gently pipette up and down with a p1000 pipette using a low retention filtered tip. The time in the dissociation buffer is critical. See image for how to assess the timing is correct by looking at your nuclei.
Image from different dissociation trials in Atlantic salmon tissues x40 magnification stained with trypan blue. A. Head kidney nuclei not had sufficient time in dissociation buffer, will clog microfluidic device. B. Blood nuclei perfectly dissociated minimal clumping ideal for sequencing. C. Liver nuclei to long in dissociation buffer, nuclear membrane started to degrade. Can still be sequenced but not ideal. Note when staining nuclei with trypan blue asses nuclear quality as soon as possible as the nuclei will quickly degrade when not on ice.
5m
Pass lysate through a 40 µm cell strainer .
Add a further 1 mL of TST to the cell strainer immediately.
Add 3 mL of freshly prepared ST buffer to the lysate.
Add the 5 mL of lysate to a marked 15 ml falcon tube (Corning) on ice.
Centrifuge at 500 x g, 4°C, 00:05:00 in a swinging bucket centrifuge.
5m
Discard liquid, carefully remove excess liquid with a p200 pipette, careful to not disturve the pellet. Resuspend the pellet gently using a p1000 pipette in diluted nuclei buffer in aiming for target recover of 6.000 nuclei. The concentration of RNAse inhibitor should be 1U/ul.
Note
Resuspension volume depends on the size of the pellet, usually within the range of 100 µL - 1000 µL (1 mL if there are many nuclei). For skin and fin, 400 µL is recommended.
Count the nuclei using a C-chip disposable haemocytometer.
Note
In this step, it is also possible to visualise the nuclei and ascertain the level of debris present as well as the integrity of the nuclear membrane.
Alternativly a non-disposable haemocytometer can be used.
The nuclei are also counted using a Bio-Rad TC20 to confirm results from the disposable haemocytometer and to count the proportion of viable cells.
Note
Nuclei are identified as “dead”, therefore a good nuclei isolation will have a small percentage of live cells. 1-4% of live cells is ideal. High proportions of live cells indicates incomplete nuclear isolation and could be an indication of high amounts of debris or insufficient lysis time.
Protocol references
Eraslan, G, Drokhlyansky E, Anand S, Subramanian A, Fiskin, E, Slyper M, Wang J, Wittenberghe N. Van; Rouhana, J.M.; Waldman, J.; et al. Single-nucleus cross-tissue molecular reference maps to decipher disease gene function. Science 2022.https://doi.org/10.1126/science.abl429090