Preparing sequencing grade RNAs from a small number of FACS-sorted larvae macrophages isolated from enzyme free dissociated zebrafish larvae

Macrophages are phagocytic cells from the innate immune system that are critical for tissue homeostasis and form the first line of host defense against invading pathogens. The zebrafish larva is an exquisite model to decipher the transcriptional response of macrophages after injury. We used a macrophage reporter line in which an mfap4 promoter drives the expression of a farnesylated mCherry fluorescent protein to label macrophages and we performed tissue dissociation, cell isolation by Fluorescence Activated Cell sorting and RNA preparation. The two bottlenecks are (i) the dissociation of the embryos that often relies on cell suspension steps that alter the activation status of immune cells, and (ii) obtaining high RNA integrity for gene expression analysis from a small number of isolated macrophages. Here, we describe (i) the dissociation of cells from whole Tg(mfap4:mCherry-F) zebrafish larvae using an enzyme-free and osmotically controlled buffer, (ii) the sorting of fluorescent macrophages by FACS and (iii) the preparation of high quality RNAs for meaningful gene expression analysis from a small number of isolated macrophages.• An optimized protocol in 5 steps to extract high quality RNAs from zebrafish macrophages.• A cell dissociation method using an enzyme-free and osmotically controlled buffer to prevent the alteration of macrophage activation status and limit cell mortality.• Production of high integrity RNAs from a small number of isolated macrophages.


a b s t r a c t
Macrophages are phagocytic cells from the innate immune system that are critical for tissue homeostasis and form the first line of host defense against invading pathogens. The zebrafish larva is an exquisite model to decipher the transcriptional response of macrophages after injury. We used a macrophage reporter line in which an mfap4 promoter drives the expression of a farnesylated mCherry fluorescent protein to label macrophages and we performed tissue dissociation, cell isolation by Fluorescence Activated Cell sorting and RNA preparation. The two bottlenecks are (i) the dissociation of the embryos that often relies on cell suspension steps that alter the activation status of immune cells, and (ii) obtaining high RNA integrity for gene expression analysis from a small number of isolated macrophages. Here, we describe (i) the dissociation of cells from whole Tg(mfap4:mCherry-F) zebrafish larvae using an enzyme-free and osmotically controlled buffer, (ii) the sorting of fluorescent macrophages by FACS and (iii) the preparation of high quality RNAs for meaningful gene expression analysis from a small number of isolated macrophages. Immunology and Microbiology More specific subject area; Establishment of immunity and inflammation, zebrafish Protocol name; A good RNA integrity from a small number of FACS-sorted macrophages for gene expression analysis Reagents/tools; All reagents and tools are described in description of protocol Experimental design; This protocol was optimized based on the Nguyen-Chi et al., publication [ 6 , 5 ].
The protocol is divided in five steps: (1) Larvae caudal fin amputation (2) Enzyme-free cell dissociation using an osmotically controlled buffer for the sorting (3) macrophage cell sorting by the FACSAria TM IIu (4) Total RNA extraction from a small number of sorted macrophages (5)  This protocol is useful for all those interested in preparing RNAs from small numbers of FACS-sorted zebrafish macrophages since the less material, the more difficult it is to obtain high quality RNAs compatible with transcriptomics analysis. Contrarily to most protocols to dissociate larvae that use enzymes (collagenase, trypsin) [ 4 , 2 , 1 ], we use a protocol that is enzyme free and osmotically controlled buffer, because we have shown that the use of trypsin to dissociate larvae induces high level expression of pro-inflammatory genes [5] . Declaration of interests; The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Zebrafish line and maintenance
Zebrafish ( Danio rerio ) maintenance, staging and husbandry were performed in the fish facility of LPHI (University of Montpellier) as described [8] . Males and females were kept in 3.5-L polycarbonate tanks connected to a recirculating Tecniplast system in following condition: 0.4% salinity/ 400 μS conductivity, temperature of 28 °C and a 12:12-h light: dark cycle. The fish were fed twice per day with Skreting GEMMA Micro 500. Embryos were obtained from pairs of adult fish by natural spawning. Embryos were collected and reared at 28 °C in 10-cm petri dishes (60 embryos/dish) containing about 25 ml of zebrafish water supplemented with methylene blue 0.1% (w/v), according to standard condition [3] . Until the desired stage, the zebrafish water is changed every day and for the experiments, larvae were staged according to Kimmel et al., and used from 3 to 4 days postfertilization (dpf). Experiments were performed using AB wild-type zebrafish and Tg ( mfap4: mCherry-F) ump6Tg animals referred here as Tg(mfap4:mCherry-F) [9] .

Procedure
To induce an acute inflammatory response, 360 Tg(mfap4:mCherry-F) larvae are either amputated or not amputated [ 7 , 11 ]. Caudal fin fold amputation is performed on 3 days post-fertilization (dpf) larvae. MCherry-F is used for specific labeling of the macrophages.
1. First, larvae are anaesthetized in zebrafish water supplemented with tricaine (final concentration 0.016% tricaine (MS-222)). For example, 60 larvae are placed into a petri dish filled with 25 ml zebrafish water (without methylene blue), then 400 μl of anesthesia tricaine (tricaine stock (4 mg/ml)) is added directly into the dish. 2. After approximatively 10 min, place the petri dish under a dissecting stereomicroscope, and manipulate the larvae in the center of the dish using a brush or a thin tip with an orientation allowing a lateral view. 3. Perform an amputation of the tip of caudal fin fold using a sterile scalpel blade, just posterior to the tip of the notochord with slight pressure, without hurting the notochord ( Fig. 1 ), as described in [7] . 4. Following amputation, with a transfer plastic pipet, transfer the sedated amputated larvae into a new petri dish filled with fresh tricaine free zebrafish water, to allow recovering of the larvae at 28 °C until the desired time points post-amputation.   9. After sorting, correctly close the cap of the collecting tube and vortex vigorously (2 × 5 s). Put the tube quickly on ice to process total RNA extraction immediately with RNeasy micro kit. Alternatively, tubes can be stored at -80 °C for a long-term storage, until extraction of total RNA with RNeasy micro kit.

Enzyme-free cell dissociation from whole wild-type AB and
Total RNA extraction from a small number of mCherry-F positive sorted macrophages from amputated and non-amputated larvae (step4) Notes: 3. According to the manufacturer's instructions, add exactly the same volume of 70% ethanol to the final volume of sorted sample, and mix immediately. As an example, 57,0 0 0 mCherry-F positive macrophages sorted into 100 μl RLT/ βME Qiagen's buffer lead to a final volume of 200 μl; the total volume of 70% ethanol that needs to be added is therefore 200 μl. 4. Follow the Qiagen RNeasy micro Kit procedure according to the manufacturer's instructions. 5. Spare 1.5 μl of each RNA sample into a new RNase/DNase-free 0.5 ml microfuge tubes that will be used for the qualitative assessment of total RNA using the Bioanalyzer 2100. 6. Keep the rest of RNA samples at −20 °C for a short-term storage or at −80 °C for a long-term storage.
Qualitative assessment of total RNA (step5) Notes:

Data measurement and analysis
Here the Table 1 and Fig. 5 provide data that validate the protocol. The Table 1 recapitulates the number of mCherry-F positive macrophages that were sorted using the FACSAria TM IIu in the different conditions (amputated and non-amputated). The Table 1 also shows representative concentrations and total amount of total RNA. In the course of these 4 independent replicates (amputated and non-amputated), we sorted from 34,0 0 0 to 97,0 0 0 mCherry-F positive macrophages and obtained from 15 to 50 nanograms of total RNA. The Fig. 5 shows assessment of the integrity of the extracted total RNAs. RNAs were ran on Pico gels and analyzed using the Agilent 2100 Bioanalyzer. Sample integrity of RNA can be determined using the RNA Integrity Number (RIN).  Table 1 Representative number of sorted-mCherry-F positive macrophages, total RNA concentrations and total amount of RNA. RNAs were prepared from sorted-mCherry-F positive macrophages from two conditions: amputated fin fold and nonamputated fin fold. The experiments were performed in 4 independent replicates. The 4 independent replicates (amputated and non-amputated) show that the total amount of RNA ranges from 15 to 50 nanograms for a number of mCherry-F positive macrophages ranging from 34,0 0 0 to 97,0 0 0 cells. RIN were calculated from 4 independent experiments and the corresponding electrophoregrams reveal that RINs range from 8 to 9.6. These values support the use of this protocol to obtain RNA with good integrity from a small number of FACS-sorted mCherry-F positive macrophages, allowing us to continue with transcriptomic analysis thanks to the good RNA quality control.

Conclusion
During inflammation, macrophage activation is associated with deep changes in their transcriptional profile. The zebrafish larva is becoming a popular model to study leukocytes thanks to its transparency and genetic tractability. Transcriptional profiling of macrophages in zebrafish larvae provided a valuable data resource to understand how macrophage respond to infection and inflammation [10] . However, this kind of analysis can be very challenging because the total number of macrophages per larva is low and it is difficult to obtain a high RNA integrity from a small number of isolated cells. Our objective was to optimize a protocol to obtain RNAs with a good integrity, in order to study the transcriptional programs of macrophages during inflammation in the zebrafish model. This protocol was optimized based on two publications: [ 6 , 5 ]. Contrarily to most protocols to dissociate larvae that use enzymes (collagenase, trypsin) [ 4 , 2 , 1 ], we developed an enzyme free protocol, combined with an osmotically controlled buffer (0.9X DPBS) to avoid over production of inflammatory mediators. In addition, our protocol was designed to maximize the total amount of extracted RNA and to favor good RNA quality. In addition, we demonstrated that this protocol is efficient to prepare RNAs from a small number of FACS-sorted zebrafish macrophages.
We believe that this protocol will be useful for all those interested in preparing RNAs with high RNA integrity from a small number of isolated macrophages to analyze macrophage transcriptional profiles in zebrafish.