Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

Our research aims to investigate the transcriptome exchanges occurring during gastrointestinal development and dysfunction in several fish larvae. Here, we provide a new protocol that allows isolation of different cell types within the Zebrafish intestine, which has potential applications in understanding gastrointestinal health and disease. In recent years, there has been growing interest in exploring the interaction between the anterior nervous system and other intestinal cell types during development and disease.

Understanding these complex dynamics using advanced techniques, like single cell RNA sequencing, holds the potential to reveal critical insights into anterior neuromyopathies, leading to more precise diagnostics and treatments. Using this protocol, we successfully identified various intestinal cell types, including epithelial, stromal, blood, muscle, immune cells, and even enteric neural ganglia, which have not been captured before at this amount using similar approaches. Thus, we provide a reliable technique to study the gastrointestinal composition during development and dysfunction.

Begin by obtaining the wildtype and transgenic Zebrafish eggs from the respective breeding setup. Take a 15 centimeter Petri dish containing HEPES-buffered E2 medium or E3, and collect the appropriate amount of fertilized eggs from the desired Zebrafish breeding setup. Incubate the fertilized eggs in E3 at 28 degrees Celsius with an appropriate light and dark cycle.

At one day's post-fertilization or DPF, remove any unfertilized eggs and let the fertilized ones develop until five DPF. Select the transgenic Zebrafish larvae at one DPF. Before proceeding for whole larva dissociation, anesthetize the five DPF wildtype larvae using 0.016%tricaine and transfer 30 anesthetized larvae into a Petri dish containing one milliliter of 10X trypsin-EDTA solution.

Then, using a razor blade, chop the larvae into small pieces in the Petri dish. Transfer the chopped larvae into a microcentrifuge tube with two milliliters of 10X trypsin-EDTA solution, and leave the tube on ice for three hours. Pipette the contents up and down with a P1000 pipette every hour to stimulate dissociation.

To begin, prepare a 1.8%agarose plate with HEPES-buffered E3 medium or E3.While working under a dissection microscope, place six to 10 anesthetized larvae in a row on the agarose plate. Put one insect pin on the head of each larvae. Then, remove the remaining E3 from the plate using a tissue.

Using another insect pin, isolate the intestine from a larva without disturbing any other organs. Ensure proper removal of yolk. Once isolated, inspect the intestine and remove all non-intestinal material, such as skin, fat, or liver.

Use tweezers to collect the intestine and transfer it to PBS containing 10%FCS in a microcentrifuge tube placed on ice. Immediately after the dissection of all intestines, centrifuge the microcentrifuge tube at 13, 800G for 30 seconds. Remove the supernatant, leaving about 100 microliters in the tube to prevent the intestines from drying out.

Add 500 microliters of papain solution to the intestines in the tube. Activate the papain by adding 2.5 microliters of one molar cysteine. Then, incubate the tube containing the intestines in a water bath at 37 degrees Celsius for 10 minutes.

Pipette the contents up and down halfway after five minutes to stimulate enzymatic tissue digestion. Transfer the dissociated cells into a fluorescence-activated cell sorting or FACS tube through a pre-wetted 35 micron cell strainer. Wash the strainer several times by adding 0.5 milliliters of PBS containing 10%FCS to a total wash volume of two milliliters.

Centrifuge the collected filtrate at 700G for five minutes at four degrees Celsius, and remove the supernatant. Add one microgram per milliliter DAPI to 300 microliters of PBS containing 10%FCS. Add this mixture to the pellet and resuspend to label the dead cells.

Then, incubate for five minutes on ice to allow their exclusion during subsequent FACS analysis. To perform fluorescence-activated cell sorting or FACS on the gut cells isolated from Zebrafish larvae, use the wildtype whole larvae sample to set the gates of the sorted cell population by recording 3, 000 cells on the flow cytometer. Use a 100 micron nozzle and set sort precision to four-way purity.

Put the collection tube containing 200 microliters of PBS with 5%fetal calf serum in the FACS machine. Load the cell suspension prepared from the isolated guts, and after excluding doublets and dead cells, sort live or DAPI-negative single cells into the collection tube. Keep the collection tube on ice after cell sorting.

Employing a viability check with trypan blue, count the number of cells in the suspension with a hemocytometer. The cells are now ready to be processed for single cell RNA sequencing. Use approximately 20, 000 cells per sample.