TITLE: Single Nuclei RNA Sequencing of Tendon Tissue - small tissue biopsies
AUTHORS: Adam Cribbs, Jolet Mimpen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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. </div><div class = "text-block">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.</div><div class = "text-block">The protocol used has been developed iteratively using healthy snap-frozen hamstring tendon but should be amenable for any tendon.</div><div class = "text-block">The protocol is used routinely within the tendon seed network for single nuclei experiments using drop-seq methods.</div></div>

[STEPS]
?. [Preparation]
Pre-cool all solutions and equipment (including centrifuge (+4oC), homogeniser (+4oC), dissection Tray (-20oC), scissors (-20oC) and scalpel/razor blade (-20oC)`).
All steps in the protocol should be performed on ice or in a cold room (4oC) to minimise RNA degradation
?. [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.
?. [Large tissue digestion]
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]
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)
?. [Tissue homogenisation]
Homogenise the sample using pestle A and stroke 20 times.
?. [Tissue homogenisation]
Place the homogeniser back onto ice and wait 5 mins.
?. [Tissue homogenisation]
Optional: Homogenise the sample using pestle B and stroke 20 times.
?. [Tissue homogenisation]
Filter the sample into a 50mL Falcon Tube passing through a 200uM strainer.
?. [Tissue homogenisation]
Incubate on ice for 5 mins.
?. [Tissue homogenisation]
Centrifuge the tube at 500g for 5mins at 4oC.
Centrifuge: 500 34, 5 min, 4 10
?. [Tissue homogenisation]
Remove the supernatant and resuspend in 4mL of NL2 buffer.
?. [Tissue homogenisation]
Incubate on ice for 5 mins.
?. [Tissue homogenisation]
Filter the sample into a 50mL Falcon Tube passing through a 40uM strainer.
?. [Tissue homogenisation]
Centrifuge at 500g for 5 mins at 4oC.
Centrifuge: 500 34, 5 min, 4 10
?. [Tissue homogenisation]
Discard the supernatant.
?. [Tissue homogenisation]
Resuspend pellet in 2mL of NW buffer.
?. [Tissue homogenisation]
Centrifuge at 500g for 5 mins at 4oC.
Centrifuge: 500 34, 5 min, 4 10
?. [Tissue homogenisation]
Discard the supernatant.
?. [Tissue homogenisation]
Resuspend pellet in 0.5-1mL of Cell sorting buffer.
?. [Tissue homogenisation]
Proceed to FACS sorting 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:
?. [Post-FACS purification of nuclei]
Sort nuclei into FACS tubes containing 1mL of NW buffer.
?. [Post-FACS purification of nuclei]
Centrifuge nuclei at 300g for 10 mins.
?. [Post-FACS purification of nuclei]
Resuspend in appropriate volume of NW buffer so that there is a ~1000 nuclei/uL.
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.
?. [Post-FACS purification of nuclei]
Nuclei that have been counted are now ready for loading into a 10X or drop-seq experiment, according to the manufacturers conditions.
?. [Large tissue digestion]
Add 2-4 ml of NL1 buffer (We usually start with 2ml and then add more if required)
?. [Large tissue digestion]
Scrape the tissue into a dounce homogeniser from the dissection tray.