TITLE: Preparation of Nuclei Suspension from Human Musculoskeletal Tissues
AUTHORS: Ching-Yan Chloé Yeung, Anja Jokipii-Utzon, Anders Karlsen

[DESCRIPTION]
An adapted version of the 'Frankenstein' protocol (Luciano G Martelotto 2020. ‘Frankenstein’ protocol for nuclei isolation from fresh and frozen tissue for snRNAseq. protocols.io https://dx.doi.org/10.17504/protocols.io.bqxymxpw) to prepare single nuclei suspensions from freshly frozen human musculoskeletal tissues before nuclei sorting by flow cytometry.

Protocol described for one sample.

This protocol has been used in the following available studies:
PREPRINT: Distinct myofibre domains of the human myotendinous junction revealed by single nucleus RNA-seq. Anders Karlsen et al. https://doi.org/10.1101/2022.12.16.519020



[BEFORE_START]
Pre-cool centrifuges
Obtain ice
Label and pre-chill tubes on ice

[GUIDELINES]
Avoid freeze-thawing of tissues
Keep materials and reagents chilled
Keep work space RNAse free
All pipetting steps are performed on ice

[STEPS]
SECTION: Tissue Preparation
1. Collect fresh tissue in a 50-ml tube containing 25 mL ice-cold 1X phosphate-buffered saline containing 50 U/ml penicillin and 50 µg/ml streptomycin and keep on ice for transport.

SECTION: Tissue Preparation
2. Using a scalpel, dissect the tissue as desired on ice.
 

SECTION: Tissue Preparation
3. Cut up tissue into small pieces of around 100 µm3, remove excess PBS from tissue pieces and snap freeze in cryotubes in liquid nitrogen. Store samples at -80 °C.
 

SECTION: Nuclei Wash and Suspension Buffer
6. Freshly prepare Nuclei Wash and Suspension Buffer (2% bovine serum albumin in PBS without magnesium, 2 mM MgCl2, 0.2 U/l RNA inhibitor). Keep on ice.
 

SECTION: Tissue Preparation
4. In a freezer cabinet or on dry ice, cut each piece of frozen tissue into smaller pieces using a scalpel. Transfer ~50 mg into a 2 ml screw cap tube containing homogenising beads. 
 

SECTION: Tissue Preparation
5. Return samples to storage at -80 °C until ready for homogenisation.

SECTION: Tissue Homogenisation
7. Pipette 1 mL chilled  to each 2 ml screw cap tube containing ~50 mg cut up frozen tissue and homogenising beads.
SECTION: Tissue Homogenisation
8. Homogenise the tissues at 4.0 M/S for 20 s in a FastPrep 24. Immediately after, transfer the tubes and incubate on ice.

SECTION: Tissue Homogenisation
9. Repeat homogenisation step. Immediately after, transfer the tubes and incubate on ice for the bubbles to settle.
 

SECTION: Tissue Homogenisation
10. Transfer the homogenate from each tube to a pre-chilled 2 ml microcentrifuge tube and add 500 µL chilled  to each tube. Mix gently using a wide bore 1000 µl pipette. Incubate the tubes on ice and gently mix two more times using a wide bore 1000 µl pipette to help release more nuclei from the remaining tissue.
SECTION: Tissue Homogenisation
11. Place a 70 µm cell strainer over a pre-chilled 15 ml tube. Filter all the homogenate from the same sample. After, wash the cell strainer with 1.5 mL chilled . Centrifuge at 500 x g, 5 min, 4 °C.

SECTION: Tissue Homogenisation
12. Remove as much supernatant as possible without disturbing the pellet. Pipette 1.5 mL chilled to the tube. Using a wide bore 1000 µl pipette gently resuspend the pellet by pipetting up and down 10 times. Transfer the suspension to a pre-chilled 2 ml microcentrifuge tube. Centrifuge at 500 x g, 5 min, 4 °C.
SECTION: Tissue Homogenisation
13. Remove as much supernatant as possible without disturbing the pellet. Pipette 500 µl chilled Nuclei Wash and Suspension Buffer without disturbing the pellet. Incubate on ice.
SECTION: Tissue Homogenisation
14. Add 1 mL Nuclei Wash and Suspension Buffer. Resuspend the pellet using an uncut 1000 µl pipette tip, gently pipetting up and down 20 times.
SECTION: Tissue Homogenisation
15. Place a 40 µm cell strainer over a pre-chilled 2 ml microcentrifuge tube. Filter the nuclei suspension. After, wash the cell strainer with 0.5 mL chilled Nuclei Wash and Suspension Buffer. Centrifuge 500 x g, 5 min, 4 °C and resuspend the pellet in the desired volume of Nuclei Wash and Suspension Buffer. Keep samples on ice.

SECTION: Nuclei Visualisation and Counting
16. Mix a small volume nuclei suspension with Trypan blue. E.g. 10 µl of each. Visualise on a Neubauer Improved Haemocytometer.
 



21. 
21. 
SECTION: Nuclei purification
17. To purify the nuclei suspension from tissue debris, e.g., collagen extracellular matrix in tendon samples, samples may be further purified by fluorescence-activated nuclei sorting or by ultracentrifugation using an iodixanol gradient (DOI: 10.1126/sciadv.abn836).