TITLE: L3 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 L3 stage C. elegans. This protocol utilizes treatment with SDS-DTT solution and Pronase E to disrupt the cuticle. Worms are mechanically homogenized with a dounce homogenizer.

[STEPS]
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.

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.

SDS-DTT solution:
To ensure treatment efficacy, the SDS-DTT solution is prepared fresh on the day of the experiment. Additionally, several aliquots of DTT powder in 1.5 ml tubes are prepared in advance and stored at -20ºC. 
To prepare the SDS-DTT solution, first resuspend 154 mg of DTT powder with 1 mL of Egg Buffer on the day of the experiment. Then, combine the following reagents in a 15 ml tube (final concentrations in parenthesis): 100 uL of 1M HEPES pH8 (20 mM), 125 uL of 10% SDS (0.25%), 1 mL of 1M DTT (200 mM), 250 uL of 60% sucrose (3%), and 3.525 mL of molecular grade water for a final volume of 5 mL.
The SDS-DTT solution is one of the most important reagents to ensure successful dissociation of post-embryonic worms. To check that the SDS-DTT solution is working properly, incubate worms in freshly prepared SDS-DTT solution under a dissection microscope. When prepared properly, worms should dissolve within ~10 minutes of observation.
SECTION: Before beginning
2. On day of protocol:

Cool swinging bucket centrifuge to 4ºC
Thaw pronase aliquots at room temperature
Place L15-10 and egg buffer on ice
Wash and sterilize dounce homogenizer and pestle A (1ml H20, 1ml 70% EtOH)
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 chemical and enzymatic treatments in this protocol require an L3 pellet less than 200 uL. If L3 pellet exceeds 200 ul, utilize 2x the L3 pellet volume.
SECTION: SDS-DTT Treatment
9. Once the worm suspension is free of E. coli, centrifuge worm suspension at 2,000 rcf for 1 minute 
SECTION: SDS-DTT Treatment
10. Resuspend the worm pellet in 1 ml of M9 and transfer to a 1.5 ml tube.
SECTION: SDS-DTT Treatment
11. Pellet the worms again at 2000 rcf for 1 minute.
SECTION: SDS-DTT Treatment
12. Transfer 10 ul of L1 worm pellet to 1ml of Qiazol and store at -80°C for downstream RNA analysis
SECTION: SDS-DTT Treatment
13. Resuspend the worm pellet in 200 ul of fresh SDS-DTT solution
SECTION: SDS-DTT Treatment
14. Incubate for 2 min room temperature
NOTE: 
- Worms should look ruffled, and pharynx should change from thin and elongated to short and round
- Incubation exceeding 2 min will damage the sample
SECTION: SDS-DTT Treatment
15. Quench reaction by adding 800 ul of ice cold egg buffer
SECTION: SDS-DTT Treatment
16. Pellet worms at 13,000 rcf for 15 seconds at 4ºC
SECTION: SDS-DTT Treatment
17. Decant the supernatant and replace with 1 ml of ice-cold egg buffer
SECTION: SDS-DTT Treatment
18. Repeat the egg buffer wash for a total of 5 washes
SECTION: Pronase E Treatment
19. Resuspend the SDS-DTT treated worms in 200 ul of 15 mg/ml Pronase E
SECTION: Pronase E Treatment
20. Incubate for 30 minutes at room temperature nutating to digest the cuticle
SECTION: Cell dissociation
27. Perform 100 strokes with dounce pestle A to generate a worm slurry
SECTION: Cell dissociation
28. Visually confirm worm dissociation by viewing a 2 ul sample of worm slurry on a fluorescent microscope
SECTION: Cell dissociation
29.2. Pellet undissociated worms at 20 rcf for 1 minute at 4ºC in swinging bucket centrifuge

NOTES:
- This step will separate the dissociated cells from intact worms
- Cells will remain in the supernatant
- Intact and partially dissociated worms will remain in the pellet
- 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.
SECTION: Measure approximate cell concentration
33. Load 10 ul of cell suspension to a Bio-Rad TC20 automated cell counting slide. Also can use a hemocytometer.
SECTION: Measure approximate cell concentration
35. Dilute the sample to    if above this concentration with L15-10
SECTION: Measure approximate cell concentration
36. Microscopically confirm fluorescent cells are present in the cell counting slide
SECTION: Measure approximate cell concentration
34. This protocol should yield between   to    total cells
SECTION: Measure approximate cell concentration
37. If the total cell yield is less than    cells and many intact worms remain, repeat the SDS-DTT, Pronase E and dounce homogenization steps
SECTION: Pronase E Treatment
21. Quench the Pronase E treatment by adding 1 ml of L15-10
SECTION: Pronase E Treatment
22. Pellet the worms at 13,000 rcf for 15 seconds at 4°C
SECTION: Pronase E Treatment
23. Decant the supernatant and resuspend the worms in 1 ml of L15-10
SECTION: Pronase E Treatment
24. Pellet the worms at 13,000 rcf for 15 seconds at 4°C
SECTION: Pronase E Treatment
25. Resuspend the worms in 1ml of L15-10
SECTION: Cell dissociation
29.3. Aspirate 1 ml of the cell-containing supernatant. Keep the pipette away from the pelleted worm debris.
SECTION: Cell dissociation
29.4. Dispense the cell suspension though a 35-micron nylon mesh filter into a 5 ml flow cytometry tube
SECTION: Cell dissociation
31. For the sorting strain, perform an additional round of dissociation and cell harvest (Steps 26-30). You will perform a total of two to three homogenization cycles, until few intact worms remain.

Total cell suspension volumes:
- Control strain = 1ml
- Sorting strain = 2-3ml
SECTION: Cell dissociation
32. - Transfer 70 ul of cells to 1 ml of Qiazol and store at -80°C for downstream RNA analysis
- Continue to step 31
- Retain the remaining ~2ml of cells for FACS
SECTION: Harvest L3 worms
4. Harvest the synchronized L3 worms by washing the plates with fresh M9
SECTION: Harvest L3 worms
5. Pass the worm suspension through a 20 micron filter. All L3 stage worms will be retained on the filter, while any contaminating worms younger than the L3 stage will pass through the filter. This step will also quickly remove a majority of contaminating E. coli. 
SECTION: Harvest L3 worms
6. Invert the filter and collect the retained L3 worms into a fresh 50 ml tube
SECTION: Harvest L3 worms
7. Transfer the filtered L3 worms to a 15 ml tube
SECTION: Harvest L3 worms
8. Pellet the worms in a 15 ml tube for 1 min at 2,000 rcf. Discard the supernatant and repeat M9 washes until the supernatant is free of visible E. coli.
SECTION: Measure approximate cell concentration
38. Move on to FACS protocol
SECTION: Cell dissociation
29. Harvest the cells
SECTION: Cell dissociation
29.1. Transfer worm slurry to 1 ml centrifuge tube
SECTION: Cell dissociation
30. Resuspend remaining worm slurry in 1ml of L15-10
SECTION: Cell dissociation
26. Transfer the worm suspension to a 2 ml glass dounce homogenizer