Preparation of Nuclei Suspension from Human Musculoskeletal Tissues
Chloé Yeung, Anja Jokipii-Utzon, Anders Karlsen
Abstract
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
Steps
Tissue Preparation
Collect fresh tissue in a 50-ml tube containing 25mL ice-cold 1X phosphate-buffered saline containing 50 U/ml penicillin and 50 µg/ml streptomycin and keep On ice for transport.
Using a scalpel, dissect the tissue as desired On ice.
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.
In a freezer cabinet or on dry ice, cut each piece of frozen tissue into smaller pieces using a scalpel. Transfer ~50mg into a 2 ml screw cap tube containing homogenising beads.
Return samples to storage at -80°C until ready for homogenisation.
Nuclei Wash and Suspension Buffer
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.
Tissue Homogenisation
Pipette 1mL chilled
Homogenise the tissues at 4.0 M/S for 0h 0m 20s in a FastPrep 24. Immediately after, transfer the tubes and incubate On ice.
Repeat homogenisation step. Immediately after, transfer the tubes and incubate On ice for the bubbles to settle.
Transfer the homogenate from each tube to a pre-chilled 2 ml microcentrifuge tube and add 500µL chilled On ice and gently mix two more times using a wide bore 1000 µl pipette to help release more nuclei from the remaining tissue.
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 1.5mL chilled500x g,4°C.
Remove as much supernatant as possible without disturbing the pellet. Pipette 1.5mL chilled500x g,4°C.
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.
Add 1mL Nuclei Wash and Suspension Buffer. Resuspend the pellet using an uncut 1000 µl pipette tip, gently pipetting up and down 20 times.
Place a 40 µm cell strainer over a pre-chilled 2 ml microcentrifuge tube. Filter the nuclei suspension. After, wash the cell strainer with 0.5mL chilled Nuclei Wash and Suspension Buffer. Centrifuge 500x g,4°C and resuspend the pellet in the desired volume of Nuclei Wash and Suspension Buffer. Keep samples on ice.
Nuclei Visualisation and Counting
Mix a small volume nuclei suspension with Trypan blue. E.g. 10 µl of each. Visualise on a Neubauer Improved Haemocytometer.
Nuclei purification
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). DOI: 10.1126/sciadv.abn836).
