Aug 13, 2023

Public workspaceNuclei Isolation for 10x Chromium single-nuclei RNA sequencing

DOI
dx.doi.org/10.17504/protocols.io.bp2l6xx4klqe/v1
  • 1Florey Institute of Neuroscience and Mental Health
courtney.wright Wright
Open access
DOI: dx.doi.org/10.17504/protocols.io.bp2l6xx4klqe/v1
Protocol Citation: Chiara Pavan, Clare Parish 2023. Nuclei Isolation for 10x Chromium single-nuclei RNA sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6xx4klqe/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 13, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 86424
Keywords: ASAPCRN, 10x-Chromium, single-nuclei RNA sequencing, nuclei isolation, sn-RNA seq, stem cells transplantation, xenografts
Abstract
Single-nucleus RNA sequencing (sn-RNA seq) enables the profiling of nuclear gene expression in isolated cells. Herein, we present a step-wise protocol for single nuclei isolation from a fresh-frozen small biopsy of rat brain containing a human xenograft. The described method includes human-neuronal nuclei isolation and debris removal using fluorescent-activated nuclei sorting. The isolated nuclei were processed through the 10x Chromium Controller platform for snRNA-seq. Compared to single-cell RNA-seq (sc-RNA seq), the use of nuclei avoids dissociation-associated transcriptional artefacts and is compatible with frozen tissue.
Guidelines
General notes

Input nuclei suspension should contain >90% dead cells with intact nuclei. The presence of a high fraction of dying cells with damaged nuclei per ambient RNA and cellular debris may influence sequencing performance

Lysis
There are various types of lysis buffer that can be used to lyse the cells and release the nuclei. This very much depends on the tissue type. The basic buffer can either be hypotonic (which might help lyse the cells and release the nuclei) or isotonic (which may be more intrinsically gentle for the preparation). The detergent that is used to lyse the cells can be varied (as well as the concentration). Commonly used buffers alternative to the one we used are:

Lysis buffer NST (isotonic+NP40)
  • 10mM Tris HCl pH 7.4
  • 146mM NaCl
  • 3mM MgCl2
  • 0.1% NP40
  • 40U/ml of RNase inhibitor

Lysis buffer IST (hypotonic + NP40)
  • 10mM Tris HCl pH 7.4
  • 10mM NaCl
  • 3mM MgCl2
  • 0.1% NP40
  • 40U/ml of RNase inhibitor

For every type of tissue, the number of strokes with the dounce homogenizer needs to be calibrated. The incubation time after the dounce homogenization can also be varied. For each tissue an optimization needs to be performed.


Tubes
Nuclei as opposed to cells are problematic since they are smaller and they are more sticky because of DNA leakage and other factors. This means that there is much more significant loss after each centrifugation step. To try to minimize the loss, we have tried various methods to prevent the nuclei sticking to the sides of the tubes. One that work and we recommend is the coating of the tubes for about one hour before use with PBS+1%BSA. Recently, we have tried using polystyrene 5ml FACS tubes but we cannot comment if they are any better at reducing loss.
Materials
Material input



Reagents

Lysis Buffer

  • ReagentPBS - Phosphate-Buffered Saline (10X) pH 7.4Thermo Fisher ScientificCatalog #AM9625
  • ReagentNuclei Isolation Kit: Nuclei EZ PrepMerck MilliporeSigma (Sigma-Aldrich)Catalog #NUC-101
  • ReagentRecombinant RNAse InhibitorTakara Bio Inc.Catalog #2313A

Wash Buffer

  • ReagentPBS - Phosphate-Buffered Saline (10X) pH 7.4Thermo Fisher ScientificCatalog #AM9625
  • ReagentUltrapure BSAAmbionCatalog #AM2616
  • ReagentProtector RNase InhibitorMerck MilliporeSigma (Sigma-Aldrich)Catalog #03335399001

Staining

  • ReagentDAPI Thermo Fisher ScientificCatalog #D1306
  • ReagentPE Anti-Human Nuclear Antigen antibody AbcamCatalog #Ab215755
  • ReagentRabbit anti-NeuN antibodyAbcamCatalog #Ab104225
  • ReagentAlexa Fluor 647 AffiniPure Goat Anti-Rabbit IgG Jackson ImmunoResearch Laboratories, Inc.Catalog #111-605-144
  • ReagentTrypan Blue Stain (0.4%) for use with the Countess™ Automated Cell CounterThermo FisherCatalog #T10282


Consumables

  • DNA LoBind Tubes, 1.5 ml (Eppendorf, cat. no. 22431021)
  • 15ml Falcon tubes (Corning , cat. no. 430052)
  • MACS Smart Strainers, 30 μm (Miltenyi Biotech, cat. no. 130-098-458 )
  • Gloves (nitrile/latex, assorted manufacturers/sizes)

Equipment
Equipment
NucleoCounter®
NAME
NC-200™
TYPE
Chemometec
BRAND
NC-200
SKU
https://chemometec.com/products/nucleocounter-nc-200-automated-cell-counter/
LINK
  • Centrifuge with swinging bucket rotor for 1.5/2.0 mL microcentrifuge tubes and for 15ml Falcon tubes
  • FACS sorter (i.e. FACS Aria III) equipped with lasers to detect Alexa Fluor 647, Phycoerythrin and DAPI
  • 10ul, 20ul, 200ul and 1000ml Pipettes and filtered tips
  • Disposable serological pipettes (5ml and 10ml) and pipette guns





Safety warnings
Attention
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Preparation of reagents and materials
Preparation of reagents and materials
  1. Prepare ~Amount50 mL of ice-cold PBS and store it on ice
  2. Prepare Amount8 mL of ice-cold lysis buffer containing 0.2U/ul of RNase inhibitor by adding Amount40 µL of RNase inhibitor to Amount8 mL of Nuclei EZ-prep
  3. Prepare Amount10 mL of ice-cold wash buffer containing 1%BSA and 0.2U/ul of RNase inhibitor by adding Amount2 mL of 5%BSA and Amount50 µL of RNase inhibitor to Amount8 mL of 1xPBS
Transcardial perfusion
Transcardial perfusion
  1. Perfuse the animal with ice-cold PBS
  2. Rapidly and TemperatureOn ice , extract the brain and using fine forceps, dissect the rat striatum containing the human xenograft
  3. Immediately transfer the tissue in an ice-cold Amount1.5 mL tube and snap-freeze in in liquid nitrogen or dry ice
  4. Store the sample to Temperature-80 °C until use


Nuclei isolation
Nuclei isolation
1h 53m
1h 53m
  1. TemperatureOn ice Transfer the tissue from the Temperature-80 °C to the dounce homogenizer containing Amount1.5 mL of cold lysis buffer TemperatureOn ice
  2. Homogenize with 24 strokes TemperatureOn ice
  3. Transfer the homogenized sample to a 15ml Corning tube on ice
  4. Wash the dounce with Amount1.5 mL of lysis buffer
  5. Allow the tube to stand for Duration00:05:00 onTemperatureOn ice
  6. Centrifuge at Centrifigation500 x g for Duration00:05:00 at Temperature4 °C
  7. Remove the supernatant
  8. Wash with Amount1 mL of lysis buffer on ice
  9. Wait Duration00:05:00 on TemperatureOn ice
  10. Centrifuge at Centrifigation500 x g for Duration00:05:00 at Temperature4 °C
  11. Remove the supernatant
  12. Resuspend in Amount1 mL wash buffer (gently, without creating bubbles) TemperatureOn ice
  13. Filter with a 30um cell strainer (MACS SmartStrainers)TemperatureOn ice
  14. Wash the strainer with Amount1 mL wash buffer TemperatureOn ice
  15. Centrifuge at Centrifigation500 x g for Duration00:05:00 at Temperature4 °C
  16. Remove the supernatant
  17. Resuspend inAmount300 µL wash buffer TemperatureOn ice
  18. Count the nuclei. We recommend using both Trypan Blue to assess nuclei integrity and a fluorescent cell counter When looking at the nuclei in the counter, look at the integrity of them – how round they are with clear border and how much they are single and dispersed and not in clumps or in small groups of cells
  19. Stain using HNA-PE and NeuN-Rb (1:100 diluted in wash buffer)
  20. Incubate for Duration00:30:00 at Temperature4 °C in the dark
  21. Add secondary antibody (Rb-647, 1:200)
  22. Incubate for Duration00:20:00 at Temperature4 °C in the dark
  23. Wash with Amount1 mL of wash buffer TemperatureOn ice
  24. Centrifuge at Centrifigation500 x g for Duration00:05:00 at Temperature4 °C
  25. Wash with 1ml of wash buffer Duration00:20:00
  26. Centrifuge at Centrifigation500 x g forDuration00:05:00 at Temperature4 °C
  27. Resuspend in DAPI solution (dilute the stock of 1mg/ml in 1:1000, 1ul in 1ml)
  28. FACS sort the nuclei in Amount35 µL of wash buffer using a 70 μm nozzle, 21– 22 p.s.i. The sort should be done for HNA+ NeuN+ DAPI+ single nuclei
  29. After FACS sorting, centrifuge the nuclei Centrifigation600 x g for Duration00:08:00
  30. Carefully remove as much supernatant as possible and count in a cell counter. Again, look at the integrity of the nuclei. The nuclei should be counted twice and the average concentration calculated.
  31. The nuclei are now ready for a 10x run. In our case sample processing was performed using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomic, California, # PN-1000121) and the Chromium Controller (10X Genomics, California) per manufacturer's instructions as published in User Guide CG000204 Rev D (10X Genomics,California)
1h 53m
Protocol references
Staining

In the described protocol, we were interested in human neuronal nuclei isolation, thereby we decided to stain the nuclei for a human (HNA) and a neuronal (NeuN) marker. This step can be avoided if the cell types of interest are different i.e. use of GFAP if the cells of interest are astrocytes. DAPI counter-staining and sorting for single nuclei is not necessary and other methods exist to remove cell debris. In our hands, the use of DAPI and sorting allowed to achieve the cleanest sample and the performance was superior compared to the use of myelin removal kit (Miltenyi) or sucrose gradient (as recommended by 10x in https://cdn.10xgenomics.com/image/upload/v1660261285/support-documents/CG000124_Demonstrated_Protocol_Nuclei_isolation_RevF.pdf