TITLE: Single-cell dissociation of Drosophila melanogaster pupal tarsi
AUTHORS: Ben R. Hopkins, Olga Barmina, Artyom Kopp

[DESCRIPTION]
This protocol outlines a step-by-step guide to generating single-cell suspensions of Drosophila melanogaster pupal tarsi for use in 10x single-cell transcriptome profiling. This protocol has been successfully used on male legs between 16h and 30h after puparium formation.

From an input of 65-70 first tarsal segments, we typically generate a suspension with the following metrics:

Expected volume of suspension generated: ~65ul
Expected cell concentration of suspension generated: ~1000-2000 cells/ul
Expected cell viability of suspension generated: ~98%

[STEPS]
SECTION: Collecting, sexing, and ageing pupae
1. Collect white prepupae. Individuals should meet the P1 aging criteria laid out by Bainbridge and Bownes (1981): the pupae should be white or cream coloured, have stopped moving completely, and display everted anterior spiracles. 
 

 

 

2. Identify individuals of the correct sex. Place white prepupae in a glass well with water under a dissection microscope. At this developmental stage the male gonads are visible on either side of the posterior ~1/3rd of the pupae.
3. Fold a kimwipe in half and then in half again a further two times. Place inside a Petri dish and wet the kimwipe with 500 µL of water. Then transfer the pupae from the glass well to the kimwipe using a paintbrush. Cover the Petri dish and move to an incubator set to 25 °C.
 


SECTION: De-casing pupae
4. At n - 1 hours, remove the Petri dish from the incubator. Using a wet paintbrush, gently remove the pupae from the by now dry kimwipe. Wetting the paintbrush helps to loosen the pupae from the wipe. Transfer the pupae to a dry kimwipe and gently pat them down to further dry them. Then transfer dorsal side up to a piece of sticky tape (sticky side up) that is secured to a flat surface (e.g. a piece of plastic or microscope slide).
 


SECTION: De-casing pupae
5. Using forceps, gently remove the pupal casing. Begin by removing the operculum and then cut laterally down the side of the case, working from anterior to posterior and gently peeling away the cut casing to expose the pupa. Then transfer the de-cased pupa to a kimwipe soaked through with water in a Petri dish.
 

 

SECTION: Dissecting legs
6. Add 100 µL of Dulbecco's PBS (DPBS) to a glass well on ice under a dissection scope.

  

SECTION: Dissecting legs
7. Use forceps to transfer several pupae, starting with the first that were de-cased, to the same tape-covered surface used for the de-casing. Place pupae ventral side up (i.e. legs facing up). 
SECTION: Dissecting legs
8. For each pupa, begin by pinching the  base of the abdomen to release fluid. This prevents the release of large amounts of fluid while pinching the leg, which interferes with the dissection. Gently press on the head to push a small amount of fluid out.
SECTION: Dissecting legs
9. To dissect forelegs, pull the second and third legs away from the body such that they point ~90° away from the body. This exposes the foreleg tarsus. The tibia/tarsus joint should be visible approximately in line with or just below the base of where the leg connects to the thorax (directly below the mouthparts).

 

SECTION: Dissecting legs
10. Using forceps, pinch just proximally to the joint and pull away from the body of the pupa. The tarsus should come away.
 

 


SECTION: Dissecting legs
11. Place the tarsus down on the tape and cover with 10 µL  of DPBS.

SECTION: Dissecting legs
12. Using a Micro Knife, slice at approximately the midsection of the tarsal segment adjacent to the segments that are being targeted i.e., if targeting the first tarsal segment slice at the midsection of the second tarsal segment. A slice can be made on both the proximal and distal side of the targeted region to isolate a subset of segments. After making the slice(s), use forceps to gently ease the targeted region out from the pupal cuticle.
 

 

SECTION: Dissecting legs
13. Using a freshly BSA-coated 10ul tip, pipette the first tarsal segment and transfer to the well of DPBS on ice.
 

SECTION: Dissecting legs
14. Repeat until the well contains 65-70 dissected tarsal segments. Two trained personnel working simultaneously can generally complete this within 90 min 

SECTION: Performing the dissociation
15. Using a 10ul tip, remove all of the DPBS from the well. Avoid removing dissected tissue.
SECTION: Performing the dissociation
16. Add 100 µL of dissociation buffer, which consists of 10x TrypLE with a final concentration of 2 mg/mL collagenase.

  
 
 


SECTION: Performing the dissociation
17. Cover the well with Nunc Aluminium Seal Tape and submerge inside of a bead bath at 37 °C for 35 min. 
 

 


SECTION: Performing the dissociation
18. Remove from the bead bath and then remove ~85 µL of dissociation buffer using a 10ul tip.
 


SECTION: Performing the dissociation
19. Add 50 µL of room temperature DPBS to the well.

SECTION: Performing the dissociation
20. Pipette the solution up and down 20x using a widebore, BSA-coated, lowbind tip. Aim for ~1.5 pipettes up and down per second, moving the tip around in the well to ensure an even dissociation throughout the suspension. Avoid introducing bubbles into the suspension. Use a 200ul pipette set to 40ul.
 

 

SECTION: Performing the dissociation
21. Pipette the solution up and down a further 20x using a BSA-coated, flame-rounded 200ul tip. Again, make sure to move the tip around in the well.
 

SECTION: Performing the dissociation
22. Slowly pipette the solution up and down a further three times using the same flame-rounded tip before taking up 40 µL and transferring to a 2mL low-bind Eppendorf tube on ice.

SECTION: Performing the dissociation
23. Add a further 20 µL of DPBS to the well, pipette up and down three times using the flame-rounded tip, and add 25 µL  to the same Eppendorf on ice.

SECTION: Performing the dissociation
24. Preview 10 µL of suspensions using a hemocytometer or automated cell counter. Concentrations and singlet rates should be checked against the requirements of the single-cell technology being used.