TITLE: Collecting, freezing and shipping frozen post mortem skeletal muscle  for single nuclear and spatial RNAseq
AUTHORS: Prech Uapinyoying

[DESCRIPTION]
Collecting and freezing skeletal muscle tissue from a clinical site for experimentation at a remote location can be a
logistical challenge. This protocol provides the procedures for collecting, snap freezing and shipping frozen skeletal muscle autopsy samples from the morgue to a remote laboratory site for downstream experiments requiring preserved muscle histology and high-quality RNA such as single-nuclear and spatial transcriptomics, and immunofluorescence


[GUIDELINES]
Tissue collection for this protocol needs prior approval by the users' Institutional Ethics Board or equivalent ethics committee.

[STEPS]
SECTION: Prior to freezing
1. Collect the human muscle autopsy samples, keeping them moist in transplant solution or saline, weigh and divide the muscle into 250mg pieces or less beforehand.  
 

SECTION: Freezing the muscle samples
2. Prepare several items and reagents and place on laboratory bench
SECTION: Freezing the muscle samples
2.1. Fill a foam box with dry ice. Cover with lid. 
SECTION: Freezing the muscle samples
2.2.  Fill the dewar with liquid nitrogen and cover.
SECTION: Freezing the muscle samples
2.3. Cut aluminum foil into 2cm x 3cm rectangular pieces. Loosely fold the foil into One per 250mg muscle. 
SECTION: Freezing the muscle samples
2.4. Tie the braided rope around the stainless-steel beaker so it can be safely lowered into the liquid nitrogen dewar
SECTION: Freezing the muscle samples
2.5. Label the cryotubes with sample ID, current date and other relevant information.
SECTION: Freezing the muscle samples
2.6. Cut a small hole in the top of the OCT bottle and place on bench
SECTION: Freezing the muscle samples
3. Fill 1/3 of the stainless-steel beaker with isopentane and carefully lower into the liquid nitrogen dewar. 
 

SECTION: Freezing the muscle samples
4. Add creases to the foil by pre-folding the foil rectangles into thirds, by folding the foil up along the long side using the small forceps. 
 

SECTION: Freezing the muscle samples
5. Take out one of the cut muscle pieces from the transplant solution and place onto kim wipes. 
SECTION: Freezing the muscle samples
7. Add a tiny dab of OCT into the middle/bottom of the foil trough and place the dried off muscle onto the OCT lengthwise.
 

   
SECTION: Freezing the muscle samples
None. Using the long tweezers, carefully grab the foil wrapped muscle sample by the pinched-off end and submerge the sample into the well of liquid isopentane for 20-30 seconds to snap freeze.
   
SECTION: Freezing the muscle samples
0.31. Pull the sample out of the isopentane, shake off the excess, slide into the labeled cryotube and place on dry ice.
SECTION: Freezing the muscle samples
None. Pull the beaker out of the liquid nitrogen dewar, allow it to thaw and pour the isopentane back into the bottle for reuse.
SECTION: Freezing the muscle samples
None. The snap frozen samples can be stored at -80°C.
SECTION: Freezing the muscle samples
6. Thoroughly dab and dry off the excess liquid from the muscle with additional wipes (approximately as dry as a fresh sample)
 

8. Loosely wrap the sides of the foil along the creases to form a tube around the muscle piece and pinch the foil at one of the long ends. Careful not to crush the muscle.
 

SECTION: Freezing the muscle samples
None. 
SECTION: Freezing the muscle samples
None. 
SECTION: Freezing the muscle samples
None. 
9. Using the long tweezers, carefully grab the foil wrapped muscle sample by the pinched-off end and submerge the sample into the well of liquid isopentane for 20-30 seconds to snap freeze.
   
10. Pull the sample out of the isopentane, shake off the excess, slide into the labeled cryotube and place on dry ice.
11. Repeat steps 4-10 until all muscle pieces are frozen and kept on dry ice.
12. Pull the beaker out of the liquid nitrogen dewar, allow it to thaw and pour the isopentane back into the original bottle for reuse.
13. The snap frozen samples can be stored at -80°C for further use.
SECTION: Preparing the samples for shipment
14. Prepare shipping material by obtaining an insulated foam box fitted inside a cardboard shipping box. 
SECTION: Preparing the samples for shipment
15. Fill the foam box with dry ice, place the sample tubes inside, and close the box 
SECTION: Preparing the samples for shipment
16. Close the insulated foam box and tape the outer cardboard box layer shut. Label and send it out for overnight shipping.