key: cord-0044330-jko5s6s3
authors: Cosacak, Mehmet Ilyas; Bhattarai, Prabesh; Kizil, Caghan
title: Protocol for Dissection and Dissociation of Zebrafish Telencephalon for Single-Cell Sequencing
date: 2020-05-29
journal: nan
DOI: 10.1016/j.xpro.2020.100042
sha: 3a3009da70e96f46788fb770df40853182d3f177
doc_id: 44330
cord_uid: jko5s6s3

Single-cell sequencing (sc-Seq) is a powerful tool to investigate the molecular signatures of cell types in a complex mixture of cells. A critical step in sc-Seq is preparing a single-cell suspension with a high number of viable cells. Here, we show how to dissect zebrafish telencephalon and how to dissociate it into a single-cell suspension. This is followed by flow cytometry-based sorting to enrich for neural progenitor stem cells. Our technique typically yields 70,000 live cells from one zebrafish telencephalon. For complete details on the use and execution of this protocol, please refer to Cosacak et al. (2019).

Protocol for dissection and dissociation of zebrafish telencephalon for single-cell sequencing Lead Contact *Correspondence: mehmet.cosacak@dzne.de, caghan.kizil@dzne.de SUMMARY Single-cell sequencing (sc-Seq) is a powerful tool to investigate the molecular signatures of cell types in a complex mixture of cells. A critical step in sc-Seq is preparing a single-cell suspension with a high number of viable cells. Here, we show how to dissect zebrafish telencephalon and how to dissociate it into a single-cell suspension. This is followed by flow cytometry-based sorting to enrich for neural progenitor stem cells. Our technique typically yields 70000 live cells from one zebrafish telencephalon.

For complete details on the use and execution of this protocol, please refer to (Cosacak et al., 2019) .

Note: Single cell sequencing can work on complex mixture of cells that are not labeled by any reporter. However, the use of a specific cell population marked by a reporter protein (e.g. GFP) will increase the portion of the resulting data that is useful for the experimental question of interest. For instance, while it is possible to sequence all cells in telencephalon simultaneously, a reporter line can enrich a specific or rare cell type by flow cytometry-based sorting. The advantage of using a reporter line is to enrich a particular cell population among other cell types in the same tissue. While it may be advantageous to sequence all cell types simultaneously, for financial considerations, enriching cell populations can be desirable.

Note: This protocol describes the use of a transgenic zebrafish line expressing green fluorescent protein (GFP) under the her4.1 promoter (Yeo et al., 2007) and marking the astroglia to enrich the astroglia that contain neural stem cells (Cosacak et al., 2019) . However, other reporters can also be used.

Preparations for dissection 1. Prepare 1X PBS, ice, petri dishes, scalpel, 50 mL tubes, 40-µM cell strainer, glass capillary, rotator and incubator (26.0 -28.5 °C). 2. Dilute β-mercaptoethanol (0.1% v/v) in 1X PBS 3. Prepare the glass capillary (Video 1). The orifice of the glass capillary can be adjusted by heating. The smallest orifice must not be too narrow. It should be comparable to the opening of a 200 µL pipette tip. CRITICAL: The enzymatic dissociation will be too slow or not possible without physical force. As a result, glass capillary is one of the options for dissociation. Trituration by glass capillary must be slow and gentle. The orifice of the glass capillary edge must not be smaller than the diameter of a cell, as it will cause lysis of the cells, which is strictly undesired. 

By using CVMI, one can deliver any substance into the zebrafish brain. Here, we describe how to use CVMI to deliver amyloid-β42, IL4 and PBS. This technique has been described in detail previously (Bhattarai et al., 2020; Bhattarai et al., 2016; Bhattarai et al., 2017a; Bhattarai et al., 2017b ). This step is not a requirement. Any desired solution can be injected into the adult zebrafish brain using the CVMI method. The experimental protocol and troubleshooting options for CVMI were described in detail previously (Bhattarai et al., 2016; Bhattarai et al., 2017a; Bhattarai et al., 2017b; Cosacak et al., 2019) . After the injection, wait for the desired duration to reach the experimental time point.

1. Dissolve Aβ42 and IL4 in PBS and inject into the zebrafish cerebroventricular fluid at 20 µM and 1 µM, respectively, as described before (Bhattarai et al., 2017b; Bhattarai et al., 2016b) . 2. Keep the fish in water system for 24h in 14/10 hours light/dark cycle as recommended (Alestrom et al., 2019) .

This is the main focus of this protocol. Here, we describe how to dissect the zebrafish telencephalon and dissociate it into single-cell suspension followed by flow cytometry-based sorting (from this point on: FACS). We provide important hints to enhance the dissociation and sorting.

CRITICAL: After starting the dissociation of cells, the protocol has NO STOP until encapsulation of cells and cDNA synthesis. This experiment must be planned for approximately 60 min deviation.

CRITICAL: Before you start, make sure that you will have access to the equipment at the time you need (e.g.: the flow cytometer, centrifuges or the single cell sequencing facility).

CRITICAL: It is always better to first optimize the dissociation and sorting of cells. Determine the optimum number of cells sorted and the percentage of these cells that survive after sorting (should not be less than 95%).

NOTE: Here, we describe our protocol for dissociation of the telencephalon in the adult zebrafish brain.

CRITICAL: Animals have to be euthanized according to the permission and ethical rules of local authorities.

CRITICAL: Before the step 3.b, animals must be sacrificed, and all further procedures will be performed post-operationally.

3. Dissecting Zebrafish Brain (1-2 mins per fish) (Video 3) a. Euthanize the fish according to the permissions and ethical rules of local authorities. b. Generate a slit at the caudal of the skull and remove the right flank of the skull carefully. c. Then remove the left flank carefully. d. Remove any extra tissues (fat or meninges) carefully. e. CRITICAL STEP: Cut the telencephalon at the border of the optic tectum and telencephalon. f. Lift the telencephalon. g. Olfactory bulb will be automatically released while lifting the telencephalon. If not, then remove it carefully. h. Gently transfer the telencephalon into Buffer Mix-I. i. Let the telencephalon rotate in a rotator at a temperature between 26.0 and 28.5 °C. j. Continue with the next fish as above.] 4. Cell Dissociation (90 mins) (Video 4) a. Rotate the brains for 10 minutes. b. Triturate (5 times) with a glass capillary to help physical dissociation (Repeat this step 3 times using the normal orifice size).

c. Add 20 µL of Buffer-Y mix (Video 4). CRITICAL: Glass capillary helps physical dissociation. However, if the orifice size is too small it will induce disruption of the cell. In general, liquid must pass easily through the orifice of the capillary. The tissue chunks should pass easily through the opening. If the orifice is too wide, it can be narrowed down by heating.

d. Incubate for 10 minutes. Triturate (5 times). Repeat this step 3 times, the orifice can be narrowed down as the chunks get smaller.

e. At the last step, triturate 5 times. If there are still visible chunks, incubate another 10 minutes. CRITICAL: Do not use small orifice sizes that might damage the cells (e.g.: shearing the cell membrane).

5. Straining and Pelleting the cells (30 min) (Video 5) a. Put a cell strainer (40 µm) in a 50 mL tube. b. Pass 5 mL of 4% BSA through the cell strainer for equilibration. CRITICAL: Always use BSA to prevent cell lysis or adherence of cells to the walls of the tubes. BSA prevents cellular stress and improves the viability significantly.

c. Pass all cell suspension with 1 mL pipette tips. Pipetting should be done gently and slowly. d. Pass 4 mL 1x PBS through the cell strainer to dilute the BSA to 2% BSA. e. Recycle the extra liquid from the cell strainer to elute all cells. f. Centrifuge in a benchtop centrifuge at 300 g for 10 min at +4 °C. NOTE: Another round of centrifugation can decrease the number of particles. This will speed up the FACS, as the number of superfluous events (e.g.: cell debris) will be decreased and intact cells will be enriched. NOTE: One can remove myelin by using commercial kits.

g. Remove supernatant carefully by inverting the tube. 

For the conditions described in the protocol above, we typically get 10% of cells as GFP-positive cells. In general, we can collect more than 70.000 live cells from one fish, this makes approximately 7000 GFP-positive cells. Depending on the gating properties these numbers can vary. 

Single cell sequencing is a powerful method for unprecedented analyses of transcriptomics in single cell resolution. Yet, some limitations do exist, as this technique is quite new and is constantly being improved.

Single cell sequencing takes into account the intact cells from which RNA reads are obtained. This is fundamentally different than conventional deep sequencing methods that are based on total RNA isolation where compromised cells also contribute to the sequencing. Therefore, in single cell sequencing, reads from hypothetically more sensitive cell types (e.g.: the ones that are compromised during the isolation procedure) could be underrepresented. End users must optimize the cell isolation and readings according to their needs and the cell types. In overall, an optimal sequencing depth and technical quality must be achieved in a particular sequencing experiment. This aspect is not at a universal standard yet and must be determined by individual end-users. TROUBLESHOOTING Problem:

Brain dissociation incomplete Potential Solution:

1. The enzymes P and A in Neural Dissociation Kit works better at 37 °C, it may require more time to dissociate fish cells at 26-28.5 °C. 

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The authors declare no competing interests.

2. Sometimes, there are large tissue parts that cannot be dissociated with longer incubation.This tissue may be contamination during dissecting the brain that the enzymes may not affect.3. Excess of PBS or other liquids may prevent enzymatic reactions. After dissecting the brain, the excess liquid can be removed (e.g.: with tissue paper) Problem:

Potential Solution:1. Always keep cells in BSA after centrifugations, as cells may stick to the plastic tubes with only 1xPBS. 2. Harsh trituration will cause break of cells, as a result trituration must be gentle without air bubbling. 3. Keeping cells on ice for longer time will cause cells to dye or be more sensitive to any temperature change. Try to minimize the time interval on ice to perform quick sorting. 4. Even healthy cells may be damaged by flow cytometer at high speed and voltage. Minimize the sorting speed and gating voltages. The nozzle size may also have effect on the cells, increasing the nozzle size will decrease cell death.Problem:

Potential Solution:1. In general, protocols are optimized for mouse or human cells (e.g., PMBC cells). For each cell type, the same protocol may not work. Test the protocol on new cell types or cells from a new organism on first use. 2. If the cells are too sensitive they may be disrupted before encapsulation. 3. Incubating cells on ice for longer durations may make cells sensitive to temperature changes or the stress emanating from previous steps during encapsulation. Minimizing incubation during dissociation, sorting will increase effective encapsulation.Note: Encapsulation is performed at 22-25 °C or higher temperatures. Sensitized cells on ice or a sudden temperature difference may cause cell lyse before encapsulation. This will make analysis more complicated; e.g. RNA background in droplets without any cell, RNA contamination from other cells may be amplified with the same barcodes. This may cause background sequencing reads and influences the downstream analyses. Normally the free-floating RNA has less influence on droplets with cells than expected as it is diluted all over the solution. The bigger problem is the RNA in droplets without a cell -there it generates background, which leads to a lower sequencing depth of the barcodes that really had a cell.

Increase the cycles of paired-end sequencing 5. Always optimize the cell dissociation and cell sorting steps before encapsulation. Improper cell dissociation and sorting may cause stress and cell death. 6. Keeping the cells on ice after sorting will cause cell death or sensitivity. Adding cells to encapsulation will lyse the cells before encapsulation. 7. Suggestion: After sorting the cells, keep cells on ice for 5, 10, 15 and 20 min. Then, pass cells through the flow cytometry and check the viability of the cells. This step has to be optimized for each cell type (e.g. fish cells, mouse cells). For instance, if the cell viability decreases at 15 min incubation on ice, then the time between the sorting and encapsulation must be minimized to max 15 min. Otherwise, cell lysed during encapsulation will cause contamination of RNA from other cells.• We describe the dissection of adult zebrafish telencephalon.• We detail a protocol for dissociation of telencephalon into single-cell suspension. • We describe a procedure for flow cytometry-based sorting to enrich NSPCs. • Our technique typically yields 70000 live cells from one zebrafish telencephalon.