TITLE: Mouse Liver Single-Cell Dissociation with Cold Protease
AUTHORS: Sanjay Subramanian, Stacey Huppert

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to dissociate murine liver cells at 4 °C in an effort to preserve transcriptomic expression profiles in cells to be used for single-cell RNA sequencing and single-cell ATAC sequencing. An expedited protocol length limits the time of exposure to dissociative solutions in order to retain natural cellular distribution and viability while limiting transcriptomic changes arising from injury response, especially in fragile cell populations. This protocol has been tested and works in mice aged 2-4 weeks, dissociating cells from the left lobe for single-cell RNA sequencing using the 10x Chromium system.</div><div class = "text-block">This project was supported in part by NIH P30 DK078392 (Gene Analysis Core) of the Digestive Diseases Research Core Center in Cincinnati, R01DK107553, and the Cincinnati Pediatric Cell Atlas Center funded by Cincinnati Children’s Research Foundation to Stacey S. Huppert</div></div>

[STEPS]
?. [Tissue Isolation]
Anesthetize mouse
?. [Tissue Isolation]
Cannulate the portal vein and flush 3-5mL ice cold 1x PBS through portal vein until liver tissue is sufficiently cleared of blood
[ice-cold 1x PBS]
?. [Tissue Isolation]
Switch PBS syringe out for one with Bacillus licheniformis mixture while keeping the portal vein cannulated. Ensure no bubbles are in the system and then perfuse 3-5mL ice cold 1mg/mL Bacillus licheniformis mixture through the portal vein
[ice-cold Bacillus licheniformis enzyme mix]
?. [Tissue Isolation]
Dissect out desired liver tissue and mince thoroughly with razor blade in petri dish with an additional 1mL Bacillus licheniformis mix; start timer for 1 hour
[ice-cold Bacillus licheniformis enzyme mix]
?. [Dissociation and Filtration]
Transfer tissue-enzyme slurry to 5mL capped tube and incubate, rotate, or rock at 4 °C until timer reaches 1 hour
[at 4 °C]
?. [Dissociation and Filtration]
Mince slurry again with razor blade, making sure to separate or scrape larger chunks to release the maximum number of cells
?. [Dissociation and Filtration]
Pass slurry through a 100μM filter into a 50mL conical tube
?. [Dissociation and Filtration]
Bring volume in tube up to 10mL by slowly passing ice cold Williams' E media with 20% FBS through filter to dislodge any sticking cells and stop the enzyme activity
[Williams'' E 20% FBS with slurry]
?. [Centrifugation and Resuspension]
Centrifuge cells at 250g for 5min at 4 °C
[centrifuge 250g at 4 °C]
?. [Centrifugation and Resuspension]
Discard supernatant and resuspend pellet in 5mL Williams E with 10% FBS
[resuspend Williams' E 10% FBS]
?. [Centrifugation and Resuspension]
Filter suspension through a 70μM filter into a new 50mL conical tube; rinse filter with an additional 5mL of media
[Williams' E 10% FBS]
?. [Centrifugation and Resuspension]
Repeat steps 9-10
[centrifuge 250g at 4 °C]
[resuspend Williams' E 10% FBS]
Final resuspension volume may be greater or less than stated and should be judged accordingly based on pellet size
?. [Quality Control]
Analyze cell morphology using hemocytometer and Trypan blue
Viable hepatocytes tend to take up trace amounts of Trypan blue
?. [Quality Control]
Adjust cell concentration as needed (700-1200 cells/μL for 10x Chromium system)
1000
?. [Tissue Isolation]
Open abdominal cavity and locate main trunk of portal vein. Make a small incision to allow for insertion of angled tip of cannulation apparatus without cutting all the way through portal vein
?. [Tissue Isolation]
Make incision in inferior vena cava to allow blood and perfusing solutions to flow out
?. [Tissue Isolation]
Fill syringe with 1x PBS and twist on to needle with attached cannulation apparatus. Flick syringe and push plunger to ensure 1x PBS has filled the tubing and there are no air bubbles in the syringe or tubing