Jun 30, 2023
Version 2
Single-cell dissociation of Drosophila melanogaster pupal tarsi V.2
- 1Department of Evolution & Ecology, University of California – Davis
External link: https://doi.org/10.1371/journal.pbio.3002148
Protocol Citation: Ben R. Hopkins, Olga Barmina, Artyom Kopp 2023. Single-cell dissociation of Drosophila melanogaster pupal tarsi. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9dzbmg3e/v2
Manuscript citation:
Hopkins BR, Barmina O, Kopp A (2023) A single-cell atlas of the sexually dimorphic Drosophila foreleg and its sensory organs during development. PLoS Biol 21(6): e3002148. doi: 10.1371/journal.pbio.3002148
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 25, 2023
Last Modified: June 30, 2023
Protocol Integer ID: 77594
Keywords: single-cell; tissue dissociation; scRNA-seq; Drosophila; tarsus; tarsi; pupa; pupal dissection; puparium; leg development; sensory organ; cuticle-associated cells
Funders Acknowledgement:
HFSP
Grant ID: LT000123/2020-L
NIH
Grant ID: R35 GM122592
Abstract
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%
Protocol materials
Dulbecco’s Phosphate Buffered SalineMerck MilliporeSigma (Sigma-Aldrich)Catalog #D8537
Step 6
TrypLE™ Select Enzyme (10X), no phenol redThermo FisherCatalog #A1217701
Step 16
CollagenaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #C0130
Step 16
Collecting, sexing, and ageing pupae
Collecting, sexing, and ageing pupae
35m
35m
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.
Note
Collect pupae n -1 hours before starting pupal de-casing, where n is the desired developmental timepoint for sequencing. Pupae are removed from the pupal case 1 hour before the leg dissection, hence the '-1'.
Note
Helpful, high-quality images of pupae at the correct stage can be found in Chyb and Gompel's 'Atlas of Drosophila morphology' (https://www.sciencedirect.com/book/9780123846884/atlas-of-drosophila-morphology).
Note
We aim to dissect 65-70 legs for a single-cell suspension. Collecting 40-50 pupae provides a buffer that allows for some failed dissections.
20m
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.
10m
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 .
Note
We have found that this volume of water is optimal. Too much and the pupal case softens and is hard to cut; too little and pupal development appears to be compromised. Beyond 30h dissections (36h and 40h), we have found 750 µL to be optimal.
5m
De-casing pupae
De-casing pupae
45m
45m
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).
Note
Transfer multiple pupae at a time to give them an opportunity to continue to dry while on the tape. This makes for easier de-casing.
5m
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.
Note
O'Connor et al. (2022) and Psathaki and Paulula (2022) both give helpful visual guides to performing this procedure.
Note
Line pupae dorsal-side down (to avoid water-logging the legs) on the kimwipe in the order in which they were de-cased.
40m
Dissecting legs
Dissecting legs
1h 30m
1h 30m
Add 100 µL of Dulbecco's PBS (DPBS) to a glass well on ice under a dissection scope.
Dulbecco’s Phosphate Buffered SalineMerck MilliporeSigma (Sigma-Aldrich)Catalog #D8537
1m
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).
3m
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 fluid out.
1m
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 developing mouthparts.
Note
The tibia/tarsus joint in the second leg can be exposed by pulling away the third leg following the same approach above. The joint is a little more proximal with respect to the mouthparts and falls approximately in line with the position of the first/second tarsal joint in the foreleg. The third leg tibia/tarsus joint is a little more proximal again.
2m
Using forceps, pinch at the joint and pull away from the body of the pupa. The tarsus should come away.
Note
At 16h after puparium formation, the actual tibia/tarsus joint seems to sit a little more proximally than the joint-like indentation that's visible in the cuticle. At this timepoint, the pinch should be made a little higher up to account for this. The pinch will need to be made more proximally if targeting tibia at any timepoint.
Note
If targeting the lower tarsal segments or pretarsus, it is still easiest to make the pinch at the tibia/tarsus joint but adjust the position of the slice in step 12.
2m
Place the tarsus down on the tape and cover with 10 µL of DPBS.
1m
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.
Equipment
Micro Knives - Plastic Handle
NAME
Ultra-thin dissecting blade
TYPE
Fine Science Tools
BRAND
10318-14
SKU
LINK
Note
Generally, the definition of the different tarsal segments is improved at later timepoints.
1m
Using a freshly BSA-coated 10ul tip, pipette the first tarsal segment and transfer to the well of DPBS on ice.
Note
BSA-coat a tip by pipetting up and down a solution of 1% BSA in DPBS 10 times.
1m
Repeat until the well contains 65-70 dissected tarsal segments. Two trained personnel working simultaneously can generally complete this within 01:30:00
1h 30m
Performing the dissociation
Performing the dissociation
52m 30s
52m 30s
Using a 10ul tip, remove all of the DPBS from the well. Avoid removing dissected tissue.
3m
Add 100 µL of dissociation buffer, which consists of 10x TrypLE with a final concentration of 2 mg/mL collagenase.
TrypLE™ Select Enzyme (10X), no phenol redThermo FisherCatalog #A1217701
CollagenaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #C0130
Note
The buffer can be premade and stored in individual aliquots at -20 °C .
2m
Cover the well with Nunc Aluminium Seal Tape and submerge inside of a bead bath at 37 °C for 00:35:00 .
Equipment
Lab Armor Beads
NAME
Non-uniform metal beads
TYPE
Gibco
BRAND
A1254301
SKU
LINK
Equipment
Auminum Seal
NAME
Auminum Seal
TYPE
Nunc
BRAND
232698
SKU
LINK
35m
Remove from the bead bath and then remove ~85 µL of dissociation buffer using a 10ul tip.
Note
The buffer removal is a balancing act. In theory, leaving too much buffer could interfere with the reagents used during 10x library preparation. However, after removing the first ~60ul it can be hard to remove more without losing tissue. Take this step slowly and move the tip to different regions of the well to avoid losing or disrupting the tissue.
3m
Add 50 µL of room temperature DPBS to the well.
2m
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.
Equipment
ART 200ul Wide Bore Filtered Pipette Tips
NAME
Pipette tips
TYPE
ART
BRAND
2069G
SKU
LINK
Note
BSA-coat the tip as in step 13.
15s
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.
Note
Prepare tips ahead of time by quickly passing them near a flame. Check under a microscope that the tip walls at the end are not rough but smooth and slightly rounded.
15s
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.
1m
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.
1m
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.
5m
Protocol references
1. S. P. Bainbridge, M. Bownes, Staging the metamorphosis of Drosophila melanogaster. Development. 66, 57–80 (1981).
2. J. T. O’Connor, E. K. Shannon, M. S. Hutson, A. Page-McCaw, Mounting Drosophila pupae for laser ablation and live imaging of the dorsal thorax. STAR Protoc. 3 (2022).
3. O. E. Psathaki, A. Paululat, Preparation of Drosophila Tissues and Organs for Transmission Electron Microscopy. Methods Mol. Biol. 2540, 361–385 (2022).