Mar 03, 2020
Single Nuclei RNA Sequencing of Tendon Tissue - small tissue biopsies
- 1University of Oxford
- Tendon Seed Network
External link: https://chanzuckerberg.com/science/programs-resources/humancellatlas/seednetworks/the-tendon-seed-network/
Protocol Citation: Adam Cribbs, Jolet Y Mimpen 2020. Single Nuclei RNA Sequencing of Tendon Tissue - small tissue biopsies. protocols.io https://dx.doi.org/10.17504/protocols.io.9sth6en
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: In development
We are still developing and optimizing this protocol
Created: November 26, 2019
Last Modified: March 03, 2020
Protocol Integer ID: 30259
Keywords: Tendon, single-nuclei, dronc-seq, nuclei, Tendon Seed Network
Abstract
This protocol is used the the Tendon Seed Network to extract nuclei from small tissue biopsies of Tendon tissue. For larger pieces opf tissue we use the GentleMACS protocol.
This protocol is an iteration of various nuclei extraction protocols (Martelotto, Humphreys, Macosko) developed specifically for tendon single-cell RNA-seq experiments, using drop-seq based techniques. The protocol can be used to isolate nuclei from both small (less than 3mmx3mm) pieces of tissue. The protocol has an optional (but recommended) FACS based isolation step if the sample contains lots of debris that can make nuclei counting difficult and sample blockages in the microfluidics more likely. However, this leads to significantly increased loss of nuclei so you may not have enough to perform your single-nuclei experiments at the end of the protocol.
The protocol used has been developed iteratively using healthy snap-frozen hamstring tendon but should be amenable for any tendon.
The protocol is used routinely within the tendon seed network for single nuclei experiments using drop-seq methods.
Guidelines
- This tissue has only been optimised for tendon samples.
- It is important to note that further optimisation is required for muscle tissues.
- It is important that the whole sample should be kept at 4oC throughout the protocol, this includes during the centrifugation steps.
- FACS purification of nuclei after tissue is important for maintaining good nuclei sequencing.
- Bypassing the FACS purification leads to low levels of UMIs, false high cell numbers and poor UMAP clustering.
- We do not recommend freezing the nuclei, as we have obtained our best results when the samples are directly loaded onto the drop-seq. However, we have not extensively evaluated this but we plan on evaluating this soon.
Materials
MATERIALS
Molecular Biology Grade WaterFisher ScientificCatalog #10154604
15 mL Falcon tubes
RNasin(R) Plus RNase Inhibitor, 10,000uPromegaCatalog #N2615
Dounce homogenizers Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8938-1SET
Nuclei EZ Lysis BufferMerck MilliporeSigma (Sigma-Aldrich)Catalog #N-3408
cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpackRocheCatalog #05 892 791 001
pluriStrainer 200 µmpluriSelectCatalog #43-50200
pluriStrainer 40 µmpluriSelectCatalog #43-50040
Falcon tube (50 mL)
Propidium iodide staining solutionBecton Dickinson (BD)Catalog #556463
PBSInvitrogen - Thermo Fisher
SUPERase• In™ RNase Inhibitor (20 U/μL)Thermo Fisher ScientificCatalog #cat# AM2694
Dissection kit
DAPIThermo Fisher ScientificCatalog #62248
Dissection Tray
Safety Scalpels, Scalpel No. 20Thermo FisherCatalog #22079720
Safety warnings
Watch your hands when using the scalpel/razor blade
Before start
Buffers and Solutions
Nuclear Isolation Buffer (NIB)
- 10mM Tris-HCl
- 10mM NaCl
- 3mM MgCl2
- 0.1% NP40
For 40mL mix:
- 400μL 1M Tris-HCl
- 200μL 2M NaCl
- 120μL 1M MgCl2
- 800μL 5% NP40
- 38.48 mL sterile water
Nuclear Lysis 1 (NL1) buffer
10mL of NIB buffer
1 tablet of cOmplete ULTRA
40 uL of RNasin Plus
40 uL of SUPERaseIN
Nuclear Lysis 2 (NL2) buffer
4mL of NIB Buffer
4 uL of RNasin Plus
4 uL of SUPERaseIN
Nuclei wash (NW) buffer
2mL of 1X PBS
2 uL of RNasin Plus
Cell sorting buffer
1mL of 1X PBS
1 uL of DAPI
Preparation
Preparation
Pre-cool all solutions and equipment (including centrifuge (+4oC), homogeniser (+4oC), dissection Tray (-20oC), scissors (-20oC) and scalpel/razor blade (-20oC)`).
Note
All steps in the protocol should be performed on ice or in a cold room (4oC) to minimise RNA degradation
Large tissue digestion
Large tissue digestion
Chop up tissue into 4 pieces on a cold dissection tray. Use scissors and razor blades that have been chilled to -20oC. The best approach to begin with is to use a razor blade and shave off a small piece of tissue. Then cut this tissue into smaller pieces and set aside on the dissection tray. Sometimes tendon can be very “stringy” and this may require the use of dissection scissors.
Scrape the tissue into a dounce homogeniser from the dissection tray.
Add 2-4 ml of NL1 buffer (We usually start with 2ml and then add more if required)
Pipette the tissue up and down 10 times using a wide bore P1000 tip (Slightly cut the top of the tip off a P1000).
Tissue homogenisation
Tissue homogenisation
Transfer the sample to a 7mL or smaller Dounce homogeniser. (If any sample remains in the dissection tray add NL1 buffer to this and add to dounce)
Homogenise the sample using pestle A and stroke 20 times.
Place the homogeniser back onto ice and wait 5 mins.
00:05:00
Optional: Homogenise the sample using pestle B and stroke 20 times.
Filter the sample into a 50mL Falcon Tube passing through a 200uM strainer.
Incubate on ice for 5 mins.
00:05:00
Centrifuge the tube at 500g for 5mins at 4oC.
500 x g, 4°C, 00:05:00
Remove the supernatant and resuspend in 4mL of NL2 buffer.
Incubate on ice for 5 mins.
00:05:00
Filter the sample into a 50mL Falcon Tube passing through a 40uM strainer.
Centrifuge at 500g for 5 mins at 4oC.
500 x g, 4°C, 00:05:00
Discard the supernatant.
Resuspend pellet in 2mL of NW buffer.
Centrifuge at 500g for 5 mins at 4oC.
500 x g, 4°C, 00:05:00
Discard the supernatant.
Resuspend pellet in 0.5-1mL of Cell sorting buffer.
Proceed to FACS sorting of nuclei.
FACS purification of nuclei
FACS purification of nuclei
FACS purification was performed using a Sony SH800 cell sorter. With our current laser settings we are unable to sort DAPI so have relied on DAPI staining of nuclei and sorting based on FSC and FL3. An example gate is shown below of a sample that shows a high level of debri:
Sample showing high levels of debris
Post-FACS purification of nuclei
Post-FACS purification of nuclei
Sort nuclei into FACS tubes containing 1mL of NW buffer.
Centrifuge nuclei at 300g for 10 mins.
Resuspend in appropriate volume of NW buffer so that there is a ~1000 nuclei/uL.
Note
A reliable nuclei count can be difficult, but we suggest that this is performed using trypan blue and confirmed using an automatic cell counter if available.
Nuclei that have been counted are now ready for loading into a 10X or drop-seq experiment, according to the manufacturers conditions.