Cassiopea xamachana Cellular Dissociation
Anthony Bonacolta, Victoria Sharp, Marta Mammone
Abstract
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
Steps
Dissociation
Cut the jellyfish tissue with a sterile razor to encourage permeability of reagents.
Gently wash the jellyfish tissue in 10mL for 0h 1m 0s then transfer to fresh 10mL and let incubate at Room temperature for 0h 2m 30s .
Using sterilized forceps, place the jelly tissue into a clean 15 mL tube then add 1mL on top of the jelly, or enough to submerge the tissue.
Incubate the tissue on a rocker for 0h 30m 0s at room temperature.
Pipette up and down using a wide-bore tip 10 times.
Repeat steps 4 and 5.
Add 80µLto the cell suspension to create a 8% FBS solution to halt enzyme digestion.
After the incubation period, filter the sample through a 70 µm filter Keep sample On ice moving forward.
Resuspend in 1000µL . Gently pipette up and down 10 times with a wide-bore tip to dissociate clumps.
Staining and Fixation
Add 1micromolar (µM) to cells. Incubate in the dark for 0h 30m 0s.
After the incubation period, filter the sample through a 70 µm filter
Resuspend in 500µL . Incubate for 0h 30m 0s .
Centrifuge 350x g,4°C then carefully discard the supernatant.
Re-suspend the pellet in 800µL using gentle pipetting with a wide-bore pipette then add 1µL .
FACS & Cryopreservation
Pre-chill chambers of FACS Machine to 4°C
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.
After sorting, cryopreserve by adding 10% volume and 1µL and immediately putting the sample at -80°C.
Thaw and Sample Submission
Thaw the sample On ice
Centrifuge 350x g,4°C then carefully remove the supernatant.
Re-suspend the cells in 1mL then add 1µL.
Submit to sequencing center On ice.
