TITLE: JAX-Sen: Mouse pancreas dissociation for single-cell RNA sequencing
AUTHORS: Ramalakshmi Ramasamy, Juliana Alcoforado Diniz, Patrick Fleming, Jessica Garofalo, Dylan Baker, Paul Robson

[DESCRIPTION]
These samples are part of the JAX-Sen project in the SenNeT Consortium. We aim to study and characterize senescence in the C57Bl/6 mouse pancreas. We compare young (6 months old) and old (24 months old) mouse pancreas using scRNAsequencing. This protocol describes the single-cell dissociation of the pancreas before library preparation and sequencing.

[STEPS]
SECTION: Reagents and Materials
1. 15 ml conical tubes – 4 per sample
1.5 or 2 ml Protein lobind tubes
Micro scissors/ scalpel
Ice-cold PBS
Histopaque-1077 – Sigma H8889
Wide-bore pipette tips
Collagenase solution
Wash buffer
100 µm cell strainer (Corning, 431752)
ACK lysis buffer – 2 ml/sample
SS_buffer – 5 ml/sample
40 um cell strainer
70 uM strainer
10 ml syringe and 18G needle if available


SECTION: Reagents and Materials
2. SS_buffer:

 

SECTION: Reagents and Materials
2.1. Collagenase solution: 
For each mouse, dissolve 4mg collagenase powder in 10mL serum-free Medium 199 (or MEM).  Prepare fresh daily.  Fill 5mL syringe with 3mL collagenase P (Roche Applied Science 11 249 002 001) and 15mL tube with 5mL collagenase; label tubes and store on ice until ready to use.  Note: each lot of collagenase must be tested experimentally to determine optimal working concentration.  Use 0.4mg/mL as starting point.

Wash buffer:  
      DMEM containing 20% FBS.

Stopping media: DMEM + 10% FBS + 1:100 dilution Glutamate CMRL media


SECTION: Procedure
2.2. Collect shipment from the warehouse.
SECTION: Procedure
2.3. Fill up ice buckets and prep them with reagents:
Bucket 1(small): PBS, HBSS, DMEM+FBS, SS_buffer
Bucket 2 (large): For mincing tissue
SECTION: Procedure
2.4. Confirm if the sample is cold. Take pictures.
SECTION: Procedure
2.5. Place tubes on ice and mince the tissue (<1mm) with sterile micro scissors.
SECTION: Procedure
2.6. After mincing, transfer the sample into labeled 15 ml tubes containing 5 ml of 0.4mg/ml collagenase solution. Incubate at 37°C for 10 mins.
SECTION: Procedure
2.7. Remove and shake tubes vigorously until pancreatic tissue is completely dissociated with no large chunks visible, 10-20 seconds.  Alternatively, triturate using 10 mL pipet until well-dispersed.
SECTION: Procedure
2.8. Immediately dilute digest with cold wash buffer to 15mL total volume.  Invert several times to mix.  Keep all the tubes cold.
SECTION: Procedure
2.9. Centrifuge at 300 x g, 4°C, 1 min to pellet pancreatic tissue.
SECTION: Procedure
2.10. Decant supernatant into waste beaker.  Add 12mL wash buffer and resuspend pellet by shaking or with 10mL pipette.
SECTION: Procedure
2.11. Repeat wash (steps #2.9-2.10) twice.  After the final spin, decant supernatant, then invert open tubes onto a paper towel and tap gently to remove all residual buffer.
SECTION: Procedure
2.12. Add 5mL Histopaque-1077 and gently resuspend pellet using 10mL pipette.
SECTION: Procedure
2.13. Hold tube at 45° angle and slowly layer 5mL wash buffer on top of gradient layer (10mL syringe is helpful for slowly dispensing buffer).  Do not allow buffer to mix with Histopaque!  Very carefully transfer tubes to centrifuge.
SECTION: Procedure
2.14. Turn off centrifuge brake.  Centrifuge at 300xg/4°C for 25 minutes.
SECTION: Procedure
2.15. Islets should be visible floating at interface between Histopaque and wash buffer, or slightly below.  Remove islets from interface using sterile transfer pipette and transfer to new 15mL conical tube. Centrifuge at 300xg/4°C for 25 minutes. Other cells (mostly acinar): in the pellet.


 
Split the 2 layers and treat them as separate samples going forward.

Proceed to islets protocol and acinar+ducts protocols that we use for human pancreatic cells.
SECTION: Acinars and Ducts:
2.16. Wash pellets from histopaq gradient 3x with 10 ml cold PBS
SECTION: Acinars and Ducts:
2.17. Add 3 ml TrypLE E to the pellet and incubate at 37C (waterbath), mixing with pipette approximately every 5 mins. Check at 10 mins and every 5 mins following for single cells. (Ducts should take 10-15 mins, and acinar should take slightly longer ~20 mins. But we have a mixture here, so check under the microscope and stop reaction accordingly)
SECTION: Acinars and Ducts:
2.18. Add 3X stopping media (9 ml).
SECTION: Acinars and Ducts:
2.19. Centrifuge at 1300 rpm/4°C for 2 minutes. Discard supernatant.
SECTION: Acinars and Ducts:
2.20. Wash again with 3 ml stopping media and pellet cells. Resuspend in 1 ml of stop media.
SECTION: Acinars and Ducts:
2.21. Filter through 40 uM Flowmi filter.
SECTION: Acinars and Ducts:
2.22. Count cells and proceed to Flex protocol.
SECTION: Islets:
2.23. Centrifuge the islets suspension at 130g/RT for 5 minutes.
SECTION: Islets:
2.24. Add 1.5 ml accutase, mix with pipette and incubate at 37C. Mix every 2 mins, checking at 6 mins.
SECTION: Islets:
2.25. Add CMRL media to approximately 15 ml total volume.
SECTION: Islets:
2.26. Centrifuge at 500xg/4°C for 5 mins. 
SECTION: Islets:
2.27. Resuspend in 1 ml of CMRI media.
SECTION: Islets:
2.28. Filter through 40 uM filter.
SECTION: Islets:
2.29. Count cells and proceed to Flex protocol.