TITLE: Cassiopea xamachana Cellular Dissociation
AUTHORS: Anthony Bonacolta, Victoria Sharp, Marta Mammone

[DESCRIPTION]
This protocol is to optimized to dissociate and fix Cassiopea xamachana cells for cell sorting and scRNA-seq.
The dissociation by itself results in 53-55% of viable cells.
Cells cannot be sorted without fixative, unless your machine can sort a seawater solution. Any other solution will lyse cells.

[BEFORE_START]
Treat reagents and materials with UV-light for ~15 mins before beginning protocol.

Set 15 mL and microcentrifuge  to 4° C.

Prepare Reagents:

Ca2+ Mg2+  free seawater (Roger et al. 2021)
    To 1 L Distilled Water add: 
        - 23 g NaCl
        - 0.763 g KCl
        - 3 g NaSO4
        - 0.25 g NaHCO3

- Dissociation Mix:
    To Ca2+ Mg2+ free ASW add:
        - 3.6 mg/mL Dispase II
        - 0.25 mg/mL of Liberase
        - 4% L-cysteine

- 1x PBS 0.5% bovine serum albumin (BSA)
        - Add 0.25 g to 50 mL 1x PBS.

- Fresh ACME Solution
        - 13:3:3:2 ratio of DNase/RNase-free distilled water, methanol, glacial acetic acid, and glycerol
        -Prep about 15 mL, FRESH, per sample each time

- RNAse Inhibitor
 

[GUIDELINES]
Make sure to work in an RNAse free-environment when able to. Use RNAse-ZAP or work in a UV sterilized hood if possible.

The tissue should be less than 1 cm long 

[STEPS]
SECTION: Dissociation
2. Gently wash the jellyfish tissue in 10 mL for 1 min then transfer to fresh  10 mL  and let incubate at Room temperature for 150 s .

SECTION: Dissociation
3. Using sterilized forceps, place the jelly tissue into a clean 15 mL tube then add 1 mL  on top of the jelly, or enough to submerge the tissue.

SECTION: Dissociation
8. After the incubation period, filter the sample through a    Keep sample  on ice  moving forward.

SECTION: Staining and Fixation
13. Centrifuge 350 x g, 7 min, 4 °C then carefully discard the supernatant.


SECTION: Staining and Fixation
14. Re-suspend the pellet in 800 µL using gentle pipetting with a wide-bore pipette then add 1 µL . 

SECTION: FACS &amp; Cryopreservation
15. Pre-chill chambers of FACS Machine to 4 °C 

SECTION: FACS &amp; Cryopreservation
16. Sort at the slowest rate (High-purity) with less than 50 PSI at 4 °C. Gate for Calcein Violet (450 nm) and chlorophyll autofluorescence (650-700 nm) for viable jelly cells and symbiont cells.

SECTION: FACS &amp; Cryopreservation
17. After sorting, cryopreserve by adding 10 % volume and  1 µL and immediately putting the sample at -80 °C.

SECTION: Thaw and Sample Submission
18. Thaw the sample on ice 

SECTION: Thaw and Sample Submission
19. Centrifuge 350 x g, 7 min, 4 °C then carefully remove the supernatant.
SECTION: Thaw and Sample Submission
20. Re-suspend the cells in 1 mL then add 1 µL. 
SECTION: Thaw and Sample Submission
21. Submit to sequencing center on ice.

SECTION: Dissociation
1. Cut the jellyfish tissue with a sterile razor to encourage permeability of reagents.
SECTION: Dissociation
4. Incubate the tissue on a rocker for 30 min at room temperature.

SECTION: Dissociation
5. Pipette up and down using a wide-bore tip 10 times.
SECTION: Dissociation
6. Repeat steps 4 and 5.
SECTION: Dissociation
7. Add 80 µLto the cell suspension to create a 8% FBS solution to halt enzyme digestion.

SECTION: Dissociation
9. Resuspend in 1000 µL . Gently pipette up and down 10 times with a wide-bore tip to dissociate clumps.

SECTION: Staining and Fixation
10. Add 1 micromolar (µM) to cells. Incubate in the dark for 30 min.

SECTION: Staining and Fixation
11. After the incubation period, filter the sample through a   
SECTION: Staining and Fixation
12. Resuspend in 500 µL .  Incubate for 30 min .