TITLE: Embryo stage C. elegans  dissociation for FACS isolation and RNA-seq analysis of intestine-specific cells
AUTHORS: Robert TP Williams, Erin Osborne Nishimura

[DESCRIPTION]
This protocol is for generating a single cell suspension suitable for isolation of intestine-specific cells through Fluorescence Activated Cell Sorting (FACS) from embryo stage C. elegans. This protocol utilizes treatment with Chitinase and Pronase E to disrupt the cuticle. Embryos are mechanically homogenized with 21G syringe needle.

[STEPS]
SECTION: Chitinase Treatment
4. Centrifuge embryo suspension at 2000 rcf for 1 minute in swinging bucket centrifuge

SECTION: Chitinase Treatment
8. Transfer 10 ul of embryo pellet to 1ml of Qiazol and store at -80°C for downstream RNA analysis
SECTION: Chitinase Treatment
9. Resuspend the embryo pellet from Step 7 in 0.5 ml egg buffer and 1 ml chitinase (1 U/ml)
SECTION: Chitinase Treatment
10. Incubate for 20 min rotating/nutating at room temperature
SECTION: Chitinase Treatment
5. Resuspend the embryo pellet in 1 ml of M9 and transfer to a 1.5 ml tube.
SECTION: Chitinase Treatment
6. Pellet the embryos at 2000 rcf for 1 minute in a centrifuge
SECTION: Chitinase Treatment
12. Pellet the embryos at 200 rcf for 5 min in fixed angle rotor centrifuge
SECTION: Chitinase Treatment
13. Aspirate and discard the supernatant
SECTION: Pronase treatment and dissociation
15. Attach a 21 guage 1¼ inch needle to a sterile 1 ml syringe
SECTION: Pronase treatment and dissociation
18. Quench the pronase treatment by adding 800 ul of L15-10 media to worm slurry
SECTION: Wash and harvest single cells
20. Wash away excess pronase from the worm slurry
SECTION: Wash and harvest single cells
20.2. Aspirate and discard the supernatant
SECTION: Wash and harvest single cells
20.4. Pellet the worm slurry at 500 rcf for 5 mins in swinging bucket centrifuge cooled to 4ºC
SECTION: Wash and harvest single cells
20.5. Aspirate and discard the supernatant
SECTION: Wash and harvest single cells
21.2.  Aspirate 1 ml of the cell-containing supernatant. Keep the pipette away from the pelleted worm debris.
SECTION: Wash and harvest single cells
21.3. Dispense the cell suspension though a 35-micron nylon mesh filter into a 5 ml flow cytometry tube
SECTION: Wash and harvest single cells
22. Perform an additional round of cell harvest for the sorting strain only (Step 21)
Total cell suspension volumes:
- Control strain = 1ml
- Sorting strain = 2ml
SECTION: Wash and harvest single cells
23. - Transfer 70 ul of cells to 1 ml of Qiazol and store at -80°C for downstream RNA analysis
- Continue to step 24
- Retain the remaining ~2ml of cells for FACS
SECTION: Pronase treatment and dissociation
14. Resuspend the chitinase treated embryo pellet in 200 ul pronase (15 mg/ml) and 500 ul egg buffer
SECTION: Pronase treatment and dissociation
16. Disrupt the embryo vitelline membrane and release cells by passing the embryo suspension through the needle 100 times, generating a worm slurry
SECTION: Pronase treatment and dissociation
17. Visually confirm embryo dissociation by viewing a 2 ul sample of worm slurry on a fluorescent microscope
SECTION: Before beginning
2. On day of protocol:

Cool swinging bucket centrifuge to 4ºC
Thaw pronase and chitinase aliquots at room temperature
Place L15-10 and egg buffer on ice
SECTION: Wash and harvest single cells
21.4. Pellet undissociated embryos at 100 rcf for 1 minute in swinging bucket centrifuge cooled to 4ºC
SECTION: Before beginning
1. Prepare reagents in advance


L15-10 Buffer: Mix 500 ml Leibovitz's L-15 Medium, 50 ml Fetal Bovine Serum (heat inactivated), 50 ul of 100x Penicillin-Streptomycin solution and 7.7 g sucrose. Filter with 0.2 micron pore filter. Store at 4ºC.

Egg Buffer: Mix 29.5 ml of 2M NaCl, 12 ml of 2M KCl, 1 ml of 1M CaCl2, 1 ml of MgCl2, 12.5 ml of 1M HEPES-NaOH pH 7.2 and 435 ml molecular grade water. Filter with 0.2 micron pore filter. Store at 4ºC.

Chitinase solution (1 U/ml): Dissolve 5 units of Chitinase from Streptomyces griseus (Sigma C6137-5UN) in 5 ml of Egg Buffer. Nutate the solution for approximately 10 minutes until dissolved. Prepare 1 ml aliquots in 1.5 ml tubes. Store aliquots at -20ºC.

Pronase E solution (15 mg/ml): Weigh 150 mg of Protease from Streptomyces griseus (Sigma P8811-1G) into a 15 ml tube. Dissolve the enzyme in 10 ml of Egg Buffer. Nutate the solution for approximately 10 minutes until dissolved. Prepare 1 ml aliquots in 1.5 ml tubes. Store aliquots at -20ºC.
SECTION: Before beginning
3. Starting material: 
Worm suspension in 15 ml tube (material generated from this protocol)
Strains: N2, fluorescent sorting strain

Perform this protocol on both strains in parallel

Note: The volumes for enzymatic treatments in this protocol require an embryo pellet less than 200 uL. If embryo pellet exceeds 200 ul, utilize 2x the embryo pellet volume.
SECTION: Chitinase Treatment
7. Aspirate and discard the supernatant
SECTION: Chitinase Treatment
11. Verify eggshell digestion by visualizing the chitinase treated embryos under a microscope. Early embryos should change shape, and pretzel stage embryos should release from their eggshell.
SECTION: Pronase treatment and dissociation
19. Store the sample on ice until all strains are completed
SECTION: Wash and harvest single cells
20.3. Resuspend the worm slurry in 1 ml of L15-10 media.
SECTION: Wash and harvest single cells
20.6. Resuspend the worm slurry in 1 ml of L15-10 media.
SECTION: Measure approximate cell concentration
24. Load 10 ul of cell suspension to a Bio-Rad TC20 automated cell counting slide
SECTION: Measure approximate cell concentration
25. This protocol should yield between   to    total cells

SECTION: Measure approximate cell concentration
26. Dilute the sample to    if above this concentration with L15-10
SECTION: Measure approximate cell concentration
27. Microscopically confirm fluorescent cells are present in the cell suspension
SECTION: Measure approximate cell concentration
28. Move on to FACS protocol
SECTION: Wash and harvest single cells
20.1.  Pellet the worm slurry at 500 rcf for 5 mins in swinging bucket centrifuge cooled to 4ºC 
SECTION: Wash and harvest single cells
21. Harvest the cells
SECTION: Wash and harvest single cells
21.1. Pellet undissociated embryos at 100 rcf for 1 minute in swinging bucket centrifuge cooled to 4ºC

NOTES:
- This step will separate the dissociated cells from intact embryos
- Cells will remain in the supernatant
- Ensure your cell type of interest is not lost during this step. 
- Visually confirm fluorescent cells are present in the supernatant. 
- Visually confirm fluorescent cells are not present in the pellet. 
- You may need to reduce the centrifuge speed and/or time if fluorescent cells are in the pellet of this step.