TITLE: Podocoryna ACME cell dissociations - draft v1.0, May 13 2024
AUTHORS: Michael Connelly

[DESCRIPTION]
This protocol is intended to dissociate and fix cells of the hydrozoan Podocoryna carnea for single-cell RNA sequencing on the 10X Chromium platform following a FACS sorting step. ACME-fixed Podocoryna cells maintain their morphology for use in microscopy and can also be frozen at -20C for storage and/or shipping.

[STEPS]
SECTION: Polyp ACME dissociation
7. Prepare for polyp dissection by placing a microscope slide with a Podocoryna colony in a glass dish with ~200 mL 0.2 um filtered seawater. Add menthol crystals at a concentration of ~1 g/L to relax the polyps for ~10 minutes.
SECTION: Medusa ACME dissociation
17. Collect as many medusae as possible using glass Pasteur pipettes. Place medusae in a petri dish with ~5 mL 0.2 um filtered seawater, and collect medusae to the center by gently swirling the dish in a circular motion. At least 300-500 medusae are recommended for good cell yields.
SECTION: Prepare materials and reagents
5. Prepare 20 mL of fresh ACME buffer.
13 mL DEPC H2O
2 mL glacial acetic acid
3 mL methanol
2 mL glycerol
Store in 1 mL aliquots at -20C if necessary
SECTION: Polyp ACME dissociation
9. Transfer 50 polyps to a 1.5 mL tube for each dissociation reaction. Centrifuge tubes at ~10,000 rcf for 30 sec to collect polyps at the bottom of the tube, then remove as much seawater as possible with a p1000 or p200.
SECTION: Medusa ACME dissociation
18. Transfer ~100 medusae to a 1.5 mL tube for each dissociation reaction. Centrifuge tubes at ~10,000 rcf for 30 sec to collect medusae at the bottom of the tube, then remove as much seawater as possible with a p1000 or p200.
SECTION: Cell staining and FACS
26. Stain cells in 900 uL PBS with 5 uL of wheat germ agglutinin and 1 uL of Hoechst. Vortex tube(s) and leave covered in the dark on ice for 30-60 mins. 
SECTION: Prepare materials and reagents
3. Prepare 15 mL of 4% MgCl2 and N-acetyl cysteine (N-Ac) wash buffer.
13.5 mL of 0.2 um filtered seawater
1.125 g of N-Ac powder
1.5 mL of 4M MgCl2
SECTION: Polyp ACME dissociation
8. Use a clean razor blade to separate the Podocoryna colony from the microscrope slide, removing the stolon and polyps together. Dissect relaxed polyps from the stolon using a clean microdissection blade and forceps, then collect the polyps into a petri dish use a glass Pasteur pipette. Transfer as little seawater and debris as possible. Approximately 300 polyps are recommended for good cell yields.
SECTION: Polyp ACME dissociation
11. Add 100 uL of the N-Ac wash buffer. Immediately, centrifuge tubes at ~10,000 rcf for 30 sec to collect polyps, then remove as much liquid as possible.
SECTION: Polyp ACME dissociation
12. Add 1 mL of ACME buffer to start the ACME dissociation. Allow ACME to permeate the tissues for 1 min. 

Next, begin pipetting using a wide-bore p1000 tip set to 500 uL volume. Pipette at the top of the ACME liquid, aim to create small air bubbles and a "foam" that will disrupt the tissue. Avoid pipetting polyps into the tip, as they can become lodged above where the liquid will reach. Pipette for 1 min, then rest for 30 sec on ice, repeat 10 times for a total of 15 minutes of dissociation.

Alternatively, use a vortex at max speed for 5 sec at 30 sec intervals for 15 mins. 
SECTION: Polyp ACME dissociation
14. Prepare pluriSelect cell strainers above a new 1.5 mL tube, with a 100 um strainer above a 40 um strainer. Pass 1 mL of ACME buffer containing dissociated cells through the paired strainers.
SECTION: Polyp ACME dissociation
15. Centrifuge tubes at 1,200 rcf for 5 min at 4C to collect dissociated cells, then aspirate 1 mL ACME buffer and resuspend pellet in 200 uL 1X PBS.
SECTION: Prepare materials and reagents
6. Prepare an ice cooler for the ACME dissociation steps.
SECTION: Prepare materials and reagents
2. Prepare 15 mL of 4% MgCl2 in filtered seawater.
13.5 mL of 0.2 um filtered seawater
1.5 mL of 4M MgCl2
SECTION: Polyp ACME dissociation
10. Add 500 uL of the 4% MgCl2 seawater and place tube on ice for 5-15 minutes. Centrifuge tubes at ~10,000 rcf for 30 sec to collect polyps, then remove as much liquid as possible (make a note if this relaxation step is omitted).
SECTION: Polyp ACME dissociation
13. Verify that polyp tissues have dissociated and a single-cell suspension has been achieved. Small tissue chunks are acceptable but whole polyps should not be visible. 

At this point, it is optional to centrifuge tubes at 1,200 rcf for 5 min at 4C to collect dissociated cells, then aspirate 1 mL ACME buffer and resuspend pellet in 200 uL 1X PBS. Place 50 uL in a well slide and view cells under a microscope to confirm dissociation quality (stain with 0.5 uL Hoechst for 30 mins if preferred).
SECTION: Polyp ACME dissociation
16. Concatenate all resuspended cells into 1-2 tubes, with 600-800 uL of cells per tube. Centrifuge tube(s) at 1,200 rcf for 5 min at 4C, aspirate supernatant and resuspend pellet in 900 uL 1X PBS. Proceed directly to cell staining and FACS steps.
SECTION: Cell staining and FACS
27. Centrifuge tube(s) at 1,200 rcf for 5 min at 4C, aspirate supernatant and resuspend pellet in 450 uL 1X PBS.
SECTION: Cell staining and FACS
28. Place 50 uL from each tube in a well slide and view cells under a fluorescent microscope to confirm dissociation and staining quality. Take photos to document cell morphology and viability, if possible.
SECTION: Cell staining and FACS
29. If satisfied with dissociated cells, combine into a single 5 mL coated FACS tube (~800 uL if using 2 tubes) and proceed to FACS sorting. Sort cells into a 5 mL coated FACS tube with 100 uL 1X PBS. 
SECTION: Cell staining and FACS
30. To collect sorted cells, centrifuge FACS tube at 1,200 rcf for 10 min at 4C, aspirate supernatant and resuspend pellet in 900 uL 1X PBS. Add 100 uL DMSO (10%, adjust volume as necessary) and freeze at -80C. 
SECTION: Prepare materials and reagents
4. Prepare FACS tubes for cell sorting by coating with 5% BSA in 1X PBS. Only empty tubes of coating solution immediately prior to sorting, do not allow them to dry out. For 50 mL 5% BSA in PBS, add 2.5 g BSA to 50 mL tube with 40 mL PBS, dissolve for 10 mins at 4C, then fill to 50 mL total.
SECTION: Medusa ACME dissociation
19. Add 500 uL of the 4% MgCl2 seawater and place tube on ice for 5-15 minutes. Centrifuge tubes at ~10,000 rcf for 30 sec to collect medusae, then remove as much liquid as possible (make a note if this relaxation step is omitted).
SECTION: Medusa ACME dissociation
20. Add 100 uL of the N-Ac wash buffer. Immediately, centrifuge tubes at ~10,000 rcf for 30 sec to collect medusae, then remove as much liquid as possible.
SECTION: Medusa ACME dissociation
21. Add 1 mL of ACME buffer to start the ACME dissociation. Allow ACME to permeate the tissues for 1 min. 

Next, begin pipetting using a wide-bore p1000 tip set to 500 uL volume. Pipette at the top of the ACME liquid, aim to create small air bubbles and a "foam" that will disrupt the tissue. Avoid pipetting medusae into the tip, as they can become lodged above where the liquid will reach. Pipette for 1 min, then rest for 30 sec on ice, repeat 10 times for a total of 15 minutes of dissociation. STOP after 4 or more rounds if tissues appear completely dissociated!

Alternatively, use a vortex at max speed for 5 sec at 30 sec intervals for 15 mins. 
SECTION: Medusa ACME dissociation
22. Verify that medusae have dissociated and a single-cell suspension has been achieved. Small tissue chunks are acceptable but whole polyps should not be visible. 

At this point, it is optional to centrifuge tubes at 1,200 rcf for 5 min at 4C to collect dissociated cells, then aspirate 1 mL ACME buffer and resuspend pellet in 200 uL 1X PBS. Place 50 uL in a well slide and view cells under a microscope to confirm dissociation quality (stain with 0.5 uL Hoechst for 30 mins if preferred).
SECTION: Medusa ACME dissociation
23. Prepare pluriSelect cell strainers above a new 1.5 mL tube, with a 100 um strainer above a 40 um strainer. Pass 1 mL of ACME buffer containing dissociated cells through the paired strainers.
SECTION: Medusa ACME dissociation
24. Centrifuge tubes at 1,200 rcf for 5 min at 4C to collect dissociated cells, then aspirate 1 mL ACME buffer and resuspend pellet in 200 uL 1X PBS.
SECTION: Medusa ACME dissociation
25. Concatenate all resuspended cells into 1-2 tubes, with 600-800 uL of cells per tube. Centrifuge tube(s) at 1,200 rcf for 5 min at 4C, aspirate supernatant and resuspend pellet in 900 uL 1X PBS. Proceed directly to cell staining and FACS steps.
SECTION: Prepare materials and reagents
1. Wear gloves for each step of protocol, clean bench spaces with RNase away and 70% ethanol prior to start.