Dec 14, 2023
Version 4
A versatile nuclei extraction protocol for single nucleus sequencing in non model species – optimization in various Atlantic salmon tissues. V.4
Peer-reviewed method
- Rose Ruiz Daniels1,
- Richard S Taylor1,
- Ross Dobie2,
- Sarah Salisbury1,
- Emily Clark1,
- Dan Macqueen1,
- Diego Robledo1
- 1The Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Edinburgh EH25 9RG, UK;
- 2Centre for Inflammation Research, The Queen’s Medical Research Institute, Edinburgh BioQuarter, University of Edinburgh, EH16 4TJ, UK
Protocol Citation: Rose Ruiz Daniels, Richard S Taylor, Ross Dobie, Sarah Salisbury, Emily Clark, Dan Macqueen, Diego Robledo 2023. A versatile nuclei extraction protocol for single nucleus sequencing in non model species – optimization in various Atlantic salmon tissues.. protocols.io https://dx.doi.org/10.17504/protocols.io.261genwm7g47/v4Version created by Rose Ruiz Daniels
Manuscript citation:
Taylor, R, Ruiz Daniels, R, Dobie, R, Naseer, S, Clark, TC, Henderson, NC, Boudinot, P, Martin, SAM & Macqueen, D 2022, 'Single cell transcriptomics of Atlantic salmon (Salmo salar L.) liver reveals cellular heterogeneity and immunological responses to challenge by Aeromonas salmonicida', Frontiers in Immunology, pp. 1-17. https://doi.org/10.3389/fimmu.2022.984799
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: December 12, 2023
Last Modified: December 14, 2023
Protocol Integer ID: 92206
Keywords: snRNA-seq , aquaculture, non-model species, nuclei
Disclaimer
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Abstract
Single cell RNA sequencing has rapidly become a standard tool for profiling transcriptomic diversity across thousands of cells (Linnarsson and Teichmann, 2016), and is now being applied to a large diversity of species and tissues. The main limitation of this technology is that it requires the isolation of live cells from fresh tissue, severely restricting its applicability. As a result, single nuclei RNA sequencing (snRNA-seq), which consists of sequencing the RNA of only the nuclei of cells rather than of the whole cell, has been commonly adopted since it allows samples to be stored for several months prior to processing while yielding comparable results to whole cell sequencing (Kulkarni, et al., 2019; Slyper et al. 2021). A critical challenge for snRNA-seq is the successful extraction of high quality nuclei. This has spurred the recent publication of a number of dissociation protocols for nuclei extraction (Drokhlyansky et al. 2020; Eraslan et al. 2021; Melms et al 2021), however, these have largely been optimized for model species such as humans, and more and more single nuclei is being adopted in non-model species.
Here we present a robust protocol that enables the extraction of nuclei from frozen tissue adapted from those shown to work in different tissue types, such as human skin (Drokhlyansky et al. 2020; Eraslan et al. 2021; Melms et al 2021). Our protocol has been used to successfully extract nuclei from an array of different Atlantic salmon (Salmo salar) tissues including liver, skin, fin, spleen, head kidney and gill as well as in other species such as sole (Solea solea) nose and gonad, rabbit (Oryctolagus cuniculus) nasal tissue and nurse shark (Ginglymostoma cirratum) spleen. We present the protocol as applied to fin and skin as these are particularly challenging tissues to work with given their toughness and the presence of hard tissue (e.g., scales and bones), connective tissue and fat deposits. We include notes throughout the protocol so that the reader can optimise it for a variety of tissue types. While the protocol has been optimised to work with the Chromium 10x platform, the most commonly used high throughput microfluidic device, but can be used successfully for the extraction of nuclei for other platforms and applications. The aim of this protocol is to capture 7,000 nuclei per single-nuclei RNA sequencing library using the Chromium Single Cell 3’ Reagent Kits v2 or v3 (10X Genomics). Given its utility for isolating nuclei from difficult to dissociate tissue types, we anticipate that this protocol will be broadly applicable for snRNA-seq of non-model organisms and unconventional tissue types.
The last step contains a supplemental video with extra context and tips, as part of the protocols.io Spotlight series, featuring conversations with protocol authors.
Guidelines
References:
CITATION
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
Kulkarni, A.; Anderson, A.G.; Merullo, D.P.; Konopka, G. Beyond bulk: a review of single cell transcriptomics methodologies and applications. Curr. Opin. Biotechnol. 2019. https://doi.org/10.1016/j.copbio.2019.03.001.
CITATION
CITATION
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 HemacytometersVwrCatalog #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 solutionVwrCatalog #97062-820
MgCl2: Magnesium chloride solution for molecular biology (1.00 M)Sigma – AldrichCatalog #M1028
Nuclease-free water: Water for biotechnology nuclease-free sterileVwrCatalog #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 InhibitorSigma AldrichCatalog #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 | 6ul µl (240 U) | 40 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-20Sigma-aldrichCatalog #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 | 1840 µl | ||
| RNAse inhibitor | 20 µl (800 U) | 200 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.
PBS+0.02 BSA (PBS+BSA) – 1 mL :
| A | B | C | |
| Stock solution | Volume | Final concentration | |
| Ultra-pure molecular grade PBS | 1970 µl | ||
| 2% BSA ** | 20 µl | ||
| RNAse inhibitor | 10 µl | 200 Uml |
** can top this up this to 2% BSA if the cells are clumping or look degraded .RNAase inhibitor is the most critical in this step as the nuclei will be in this buffer the longest can use up to 1000 U per ml.
Protocol materials
NaCl (5 M) RNase-freeThermo Fisher ScientificCatalog #AM9759
Materials
Tungsten Carbide Straight 11.5 cm Fine Scissors Fine Science ToolsCatalog #14558-11
Materials
Protector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #3335399001
Materials
Calcium chloride 1 M in aqueous solutionVWR InternationalCatalog #97062-820
Materials
UltraPure™ 1 M Tris-HCI Buffer, pH 7.5Thermo FisherCatalog #15567027
Materials
pluriStrainer Mini 20 µm (Cell Strainer)pluriSelectCatalog #43-10020-50
Materials
Corning™ Falcon™ Test Tube with 35µm Cell Strainer Snap CapCorningCatalog #352235
Materials
Magnesium chloride solution for molecular biology (1.00 M)Merck MilliporeSigma (Sigma-Aldrich)Catalog #M1028
Materials
INCYTO C-Chip™ Disposable HemacytometersVWR InternationalCatalog #82030-468
Materials
Falcon™ Cell StrainersFisher ScientificCatalog #08-771-2
Materials
Noyes Spring Scissors - Tungsten CarbideFine Science ToolsCatalog #15514-12
Materials, Step 2
Tween-20Merck MilliporeSigma (Sigma-Aldrich)Catalog #P-7949
Materials
Water for biotechnology nuclease-free sterileVWR InternationalCatalog #97062-794
Materials
Bovine Serum Albumin (20 mg/mL) Molecular Biology GradeNew England BiolabsCatalog #B9000S
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.
Before using this prep for library preparation do a trial run.
Recommended to do a trial especially on a new tissue type to adjust different parameters without adding RNase. Once parameters are adjusted such as mincing times, filter size and dilution in to final buffer in order to get good quality nuclei.
Nucleus isolation workflow for ST-based buffers
Nucleus isolation workflow for ST-based buffers
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 CarbideVWR InternationalCatalog #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
Resuspend the pellet gently using a p1000 pipette in PBS-BSA.
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.
Filter the nucleus solution a second time.
Note
The size of the filter is tissue dependant, e.g. for tissues such as liver and head kidney a 40 µm Falcon™ cell strainer will suffice, whereas for gill, a 30 µm filter would be better giving the higher amount of tough debris that could clog the microfluidic device. In addition, for harder tissues that produce a lot of debris such as fin and skin (this is due to the presence of fat layers and scales in skin and the presence of bones in the fin) then 20 µm is recommended. The lysate may not pass through at once, pipetting very gently up and down with a wide bore pipette can help it through.
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.
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, but below 12% is acceptable. High proportions of live cells indicates incomplete nuclear isolation and could be an indication of high amounts of debris or insufficient lysis time.
Load the nucleus suspension into a Chromium Chip and into the Chromium Controller, aiming to recover 7,000 nuclei as per 10x recommendations with a concentration of between 700 to 1200 nuclei per µl.
Note
In the case of some tissues such as fin, readjust the target recovery to 5000 especially with juvenile fish or tissues such as fin and skin as nuclei yields are on the low side.
Spotlight video
Spotlight video
30m
Citations
Slyper M, Porter CBM, Ashenberg O, Waldman J, Drokhlyansky E, Wakiro I, Smillie C, Smith-Rosario G, Wu J, Dionne D, Vigneau S, Jané-Valbuena J, Tickle TL, Napolitano S, Su MJ, Patel AG, Karlstrom A, Gritsch S, Nomura M, Waghray A, Gohil SH, Tsankov AM, Jerby-Arnon L, Cohen O, Klughammer J, Rosen Y, Gould J, Nguyen L, Hofree M, Tramontozzi PJ, Li B, Wu CJ, Izar B, Haq R, Hodi FS, Yoon CH, Hata AN, Baker SJ, Suvà ML, Bueno R, Stover EH, Clay MR, Dyer MA, Collins NB, Matulonis UA, Wagle N, Johnson BE, Rotem A, Rozenblatt-Rosen O, Regev A. A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.
https://doi.org/10.1038/s41591-020-0844-1Drokhlyansky E, Smillie CS, Van Wittenberghe N, Ericsson M, Griffin GK, Eraslan G, Dionne D, Cuoco MS, Goder-Reiser MN, Sharova T, Kuksenko O, Aguirre AJ, Boland GM, Graham D, Rozenblatt-Rosen O, Xavier RJ, Regev A. The Human and Mouse Enteric Nervous System at Single-Cell Resolution.
https://doi.org/10.1016/j.cell.2020.08.003Linnarsson S, Teichmann SA. Single-cell genomics: coming of age.
https://doi.org/10.1186/s13059-016-0960-x