Laser-Assisted Microdissection and High-Throughput RNA Sequencing of the Arabidopsis Gynoecium Medial and Lateral Domains

Cite as: Luna-García, V. and de Folter, S. (2024). Laser-Assisted Microdissection and High-Throughput RNA Sequencing of the Arabidopsis Gynoecium Medial and Lateral Domains. Bio-protocol 14(17): e5056. DOI: 10.21769/BioProtoc.5056. Copyright: © 2024 The Authors; exclusive licensee Bio-protocol LLC. This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/). 1 Published: Sep 05, 2024 Open Access Laser-Assisted Microdissection and High-Throughput RNA Sequencing of the Arabidopsis Gynoecium Medial and Lateral Domains Valentín Luna-García and Stefan de Folter* Unidad de Genómica Avanzada (UGA-Langebio), Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (Cinvestav), Irapuato, Mexico *For correspondence: [email protected] Abstract Key features • Laser-assisted microdissection (LAM) allows the isolation of specific cells or tissues. • Normal slides can be used for LAM. • It allows the identification of the transcriptional profiles of specific tissues of the Arabidopsis gynoecium. Keywords: Laser-assisted microdissection, LAM, Specific cells and tissues, Gynoecium, Arabidopsis, Transcriptomes, RNA-Seq This protocol is used in: Plant Physiol (2024), DOI: 10.1093/plphys/kiad658 For obtaining insights into gene networks during plant reproductive development, having transcriptomes of specific cells from developmental stages as starting points is very useful. During development, there is a balance between cell proliferation and differentiation, and many cell and tissue types are formed. While there is a wealth of transcriptome data available, it is mostly at the organ level and not at specific cell or tissue type level. Therefore, methods to isolate specific cell and tissue types are needed. One method is fluorescent activated cell sorting (FACS), but it has limitations such as requiring marker lines and protoplasting. Recently, single-cell/nuclei isolation methods have been developed; however, a minimum amount of genetic information (marker genes) is needed to annotate/predict the resulting cell clusters in these experiments. Another technique that has been known for some time is laser-assisted microdissection (LAM), where specific cells are microdissected and collected using a laser mounted on a microscope platform. This technique has advantages over the others because no fluorescent marker lines must be made, no marker genes must be known, and no protoplasting must be done. The LAM technique consists in tissue fixation, tissue embedding and sectioning using a microtome, microdissection and collection of the cells of interest on the microscope, and finally RNA extraction, library preparation, and RNA sequencing. In this protocol, we implement the use of normal slides instead of the membrane slides commonly used for LAM. We applied this protocol to obtain the transcriptomes of specific tissues during the development of the gynoecium of Arabidopsis. Cite as: Luna-García, V. and de Folter, S. (2024). Laser-Assisted Microdissection and High-Throughput RNA Sequencing of the Arabidopsis Gynoecium Medial and Lateral Domains. Bio-protocol 14(17): e5056. DOI: 10.21769/BioProtoc.5056. 2 Published: Sep 05, 2024 Graphical overview Laser-assisted microdissection (LAM) procedure of gynoecium tissues in Arabidopsis Background Multicellular organisms, such as animals and plants, are made up of many cells, which are grouped together to form different tissues and organs. Proper development requires complex molecular regulation, ranging from changes in gene expression and signaling events to hormonal activity. Despite the efforts made to understand these mechanisms that regulate development, the great morphological complexity of tissues or organs represents a limitation to creating tissue-specific expression profiles [1–4]. However, in recent years, several techniques have been successfully developed and implemented to isolate and characterize the transcriptomic profiles of specific cell populations, such as fluorescent activated cell sorting (FACS), single-cell isolation, single-nuclei isolation, and laser-assisted microdissection (LAM) [5]. LAM is a microscopy-based technique that has established itself as a good technique for isolating and collecting specific cell populations from their tissue context [6–11]. The LAM technique is distinguished from others for not requiring the generation of marker lines or protoplasts or previous information on a large number of marker genes; it only requires knowing the morphology of the tissues to be collected. These characteristics have allowed expanding LAM implementation to different organs of different plant species such as tomato fruit [12, 13], rice [14], California poppy [15], and Arabidopsis [16–18]. Here, we describe a protocol, built on previous work by Chávez Montes et al., 2016 [10], to perform LAM and RNA-seq of four tissue types from the Arabidopsis gynoecium: carpel marginal meristem (CMM) and septum (SEP), both tissues of the medial domain of the gynoecium, and the presumptive carpel wall (PC) and valves (VV), two tissues that make up the lateral domain of the gynoecium [19]. The aim was to identify the transcriptomic profiles that regulate their development. In the implementation of this protocol, the use of slides without the membrane was chosen instead of the commercially available slides (MembraneSlides) for LAM, which was better for the collection of the tissues of interest. Furthermore, we briefly describe the data analysis and gene expression visualization. This protocol should also be applicable for other plant species. Cite as: Luna-García, V. and de Folter, S. (2024). Laser-Assisted Microdissection and High-Throughput RNA Sequencing of the Arabidopsis Gynoecium Medial and Lateral Domains. Bio-protocol 14(17): e5056. DOI: 10.21769/BioProtoc.5056. 3 Published: Sep 05, 2024 Materials and reagents Biological materials 1. Arabidopsis thaliana plants (inflorescence tissue) Reagents 1. Absolute ethyl alcohol for molecular biology (ethanol) (Karal, catalog number: 2003) 2. Glacial acetic acid (Fermont, catalog number: 03015) 3. Histo-Clear II (Electron Microscopy Sciences, catalog number: 64111-04) 4. Paraplast Plus® (paraffin) (Sigma-Aldrich, catalog number: P3683) 5. Methyl alcohol (Karal, catalog number: 2010) 6. Direct-zol RNA Microprep (Zymo Research, catalog number: R2063) Solutions 1. Fixation solution (see Recipes) Note: We do not recommend using formaldehyde (FDA; paraformaldehyde) as the fixation solution, because a cross-link reversal step would be needed for RNA extraction. Some discussion on fixation solutions is given in Chávez Montes et al. [10]. Recipes 1. Fixation solution Reagent Final concentration Quantity or Volume Ethanol (absolute) 75% (v/v) 3 mL Glacial acetic acid 25% (v/v) 1 mL Total n/a 4 mL Laboratory supplies 1. Sterile falcon tubes 2. Beakers 3. Dissecting forceps 4. Steel inclusion molds 15 mm × 15 mm (Citotest) 5. Embedding cassettes without lid, color white (Simport Scientific, catalog number: M480-2) 6. Microscope slides 26 × 76 mm, thickness of 1 mm (LAUKA) 7. Low-profile disposable blades (Leica, model: 819) 8. Slide rack and glass staining dish with lid (LaboLan, catalog number: 29600001XL) 9. TubeCollector 2×200 CM II (Zeiss, catalog number: 415101-2000-410) 10. AdhesiveCap 200 clear (Zeiss, catalog number: 415190-9191-000) 11. MembraneSlide 1.0 OPEN (Zeiss, catalog number: 415101-4401-000) Equipment 1. Vacuum pump (Gast, model: DOA-P704-AA) 2. Vacuum desiccator 3. Mini-rotator, 2–80 rpm w/disk & clamps (Glas-Col, catalog number: 099A MR1512) Cite as: Luna-García, V. and de Folter, S. (2024). Laser-Assisted Microdissection and High-Throughput RNA Sequencing of the Arabidopsis Gynoecium Medial and Lateral Domains. Bio-protocol 14(17): e5056. DOI: 10.21769/BioProtoc.5056. 4 Published: Sep 05, 2024 4. Convection incubator (Binder, model: BD 23) 5. Paraffin dispenser (Leica, model: EG 1120) 6. Microtome (Leica, model: RM2035) 7. Slide warmer (Lab-Line Instruments, model: 26005) 8. PALM MicroBeam IV microscope with motorized RoboMover and CapMover (Zeiss, series number: 1214000156) 9. NanoDrop (optional) 10. Covaris S2 ultrasonicator (optional) 11. Qubit 4 fluorometer (optional) 12. Bioanalyzer 2100 (optional) Software and datasets 1. PALM Robo software (Zeiss, v4) 2. Optional: R studio for RNA-seq data analysis Procedure A. Embedding inflorescence tissues in paraffin 1. Tissue fixation a. Day 1. Collect inflorescence tissue (use dissecting forceps) and place the tissue directly into the fixation solution [ethanol: glacial acetic acid 3:1 (v/v)] on ice. Notes: i. Prepare the fixation solution fresh and keep cold. ii. As a guideline, for 15–20 Arabidopsis inflorescences in a 50 mL Falcon tube or beaker, 10 mL of each solution (for this and the next steps) is needed. The tissue should always be covered with solution. iii. Tissues of different genotypes or treatments should be maintained separated and always labeled. Caution: RNA integrity should be conserved. Work clean and use gloves. b. Apply vacuum for 15 min using a vacuum desiccator. Notes: i. The open Falcon tube or beaker is on ice in the vacuum desiccator. ii. In our work, we use the maximum force of the vacuum pump, which is around -0.6 bar. c. Replace the fixation solution and repeat the vacuum step for 15 min. d. Leave the samples in fixation solution overnight at 4 °C. Notes: i. The tissue should sink during the vacuum steps. If not, repeat vacuum step. ii. It is most practical to use a 50 mL Falcon tube(s) with cap from this point onward. 2. Dehydration a. Day 2. Dehydrate the tissue samples using five steps of ethanol concentrations (70%, 80%, 90%, 100%, 100%). For each step, incubate the samples for 1 h at 4 °C. Constant agitation of 10 rpm may be used. b. Leave samples in 100% ethanol at 4 °C overnight. Pause point: Samples may be stored at 4 °C for more days if needed. 3. Histo-Clear II incubation a. Day 3. Incubate the samples in a series of three steps with ethanol:Histo-Clear II (v/v) solutions [3:1, 1:1, 1:3 (v/v)]. For each step, incubate the samples for 1 h. Note: Histo-Clear II is a replacement for the use of xylene, which is toxic. b. Place the samples in 100% Histo-Clear II for 1 h. Cite as: Luna-García, V. and de Folter, S. (2024). Laser-Assisted Microdissection and High-Throughput RNA Sequencing of the Arabidopsis Gynoecium Medial and Lateral Domains. Bio-protocol 14(17): e5056. DOI: 10.21769/BioProtoc.5056. 5 Published: Sep 05, 2024 c. Remove half of the Histo-Clear II volume by decanting and replace this volume with paraffin wax pellets (Paraplast Plus®). Leave overnight at room temperature. Note: We recommend transferring the samples and solution to a small beaker(s), although Falcon tubes also work. 4. Paraffin incubation a. Day 4. Place the beaker(s) with samples from the previous step in an incubator at 58 °C for 15–20 min (or until the Histo-Clear and paraffin mixture becomes liquid). During this step, all paraffin wax pellets will melt. Notes: i. We recommend the use of a convection incubator. Do not leave the door open too much time to avoid a drop in temperature. The use of a bigger incubator helps to maintain the temperature. ii. The temperature of melting the paraffin is crucial and must be maintained according to the brand of paraffin in use. b. Once the mixture is homogenized, decant a quarter of the total volume and replace this same volume with liquid paraffin at 58 °C. Incubate for 1 h at 58 °C in the incubator. Note: For a correct homogenization of liquid paraffin, the paraffin dispenser should be turned on between 1 and 2 days prior to use; this depends on the type of paraffin dispenser and the amount of paraffin it contains. If no paraffin dispenser is available, Falcon tubes with melted paraffin stored at 58 °C may be used; these can be in the same incubator. c. After 1 h, decant half of the total volume, replace this same volume with liquid paraffin at 58 °C, and place immediately back in the incubator at 58 °C for 1 h. d. After 1 h, decant three-quarters of the total volume and replace this same volume with liquid paraffin at 58 °C. Incubate for 1 h at 58 °C in the incubator. Note: From this step onward, the mixture is likely to start solidifying faster than in the previous steps; therefore, the work should be done as fast as possible to avoid solidification. e. In the fourth wash, decant the whole mixture and replace it with liquid paraffin. Incubate again for 1 h at 58 °C in the incubator. Additional time may be needed depending on whether the paraffin is completely liquid; if not, incubate longer. f. Repeat step A4e once or twice. If the smell of Histo-Clear II is no longer perceived, it is not necessary to continue with paraffin replacements. g. After paraffin replacement steps, leave the samples in paraffin and incubate at 58 °C overnight in the incubator. 5. Embedding the tissue in paraffin blocks a. Day 5. Prepare the paraffin blocks with the tissue (Figure 1). First, remove the beaker with inflorescences from the incubator and transfer the tissue with liquid paraffin to a hot plate (~58 °C), to maintain the liquid state of the paraffin. Note: Some paraffin dispensers come coupled with a plate, but if you do not have this model, you can use any other hot plate. Practical tip: The plate can be covered with aluminum foil for easier cleaning afterward. b. Place the steel inclusion molds also on top of the hot plate (~58 °C) to warm them; this helps for better handling of the melted paraffin during the generation of the blocks. c. Next, add a small amount of liquid paraffin to the well of the inclusion mold. Subsequently, place the inflorescences (from 1 to 4 inflorescences per block) in the position you want to obtain the histological section (transverse or longitudinal). Then, place the plastic embedding cassette on top of the mold and fill the mold completely with liquid paraffin, let it cool, and then let it solidify on ice. See Figure 1 for various parts of the preparation of a paraffin block. d. Store the block(s) at 4 °C until use. Critical: Maintain the blocks free of RNase. Cite as: Luna-García, V. and de Folter, S. (2024). Laser-Assisted Microdissection and High-Throughput RNA Sequencing of the Arabidopsis Gynoecium Medial and Lateral Domains. Bio-protocol 14(17): e5056. DOI: 10.21769/BioProtoc.5056. 6 Published: Sep 05, 2024 Figure 1. Overview of the complete laser-assisted microdissection (LAM) protocol. The protocol starts with plant tissue fixation, followed by tissue embedding in paraffin blocks, specific tissue/cell collection with the microscope, and finally RNA extraction, library preparation, and RNA sequencing. Taken from Luna-García et al. [19] (Supplementary Figure S1) with permission. B. Histological sections 1. Day 6. Prepare the histological sections from the paraffin blocks with the tissue of interest using a microtome. In our experience, a thickness from 8 to 12 microns works well for the microdissection of Arabidopsis gynoecium tissues (Figure 1). Notes: a. The thickness of the histological sections can vary according to the tissue and the needs of each project. b. Previous experience with a microtome is strongly advised. 2. Observe the cross sections with the help of an optical microscope; use only those sections in which the tissue of interest is present. Once the histological sections to be used have been identified, place them on a slide(s) as described in the next step. Note: Slides with a membrane (typically used) to maintain cell integrity during LAM can be used, but for Cite as: Luna-García, V. and de Folter, S. (2024). Laser-Assisted Microdissection and High-Throughput RNA Sequencing of the Arabidopsis Gynoecium Medial and Lateral Domains. Bio-protocol 14(17): e5056. DOI: 10.21769/BioProtoc.5056. 7 Published: Sep 05, 2024 the development of this protocol with Arabidopsis inflorescences, slides without membrane were used since they allowed a better manipulation during microdissection. 3. Place slides on a slide warmer at ~42 °C. Add pure methanol to each slide surface. Place/float the selected paraffin sections (ribbons of paraffin sections; Figure 1) on the methanol, remove the excess methanol, and let the slides dry for ~45–60 min on the slide warmer. This will allow the fixation of the histological sections to the slides. Notes: a. The slides with fixed sections can be used directly in the next step of deparaffinization and LAM; alternatively, the non-deparaffinized slides can be stored for 1–2 days maximum at 4 °C. b. We did not evaluate that but, when working with slides stored for longer times, the amount of extracted RNA from the LAM tissue decreased. c. We use, as others do, pure methanol on the slide surface instead of water to minimize RNase activity. d. Caution: Methanol is highly toxic. C. Laser-assisted microdissection (LAM) of medial and lateral gynoecia tissues 1. Day 7. Deparaffinize the slides by placing them in a slide rack in a glass staining dish with 100% HistoClear II for 15 min (Figure 1). Subsequently, let the slides dry at room temperature for ~20–30 min. Afterward, start with the microdissection of the tissues of interest as described in the next steps. Critical: Remember to keep everything free of RNase. 2. Laser-microdissection of tissue of interest a. Turn on the PALM microscope (Figure 2A) and open the PALM Robo v4 software (Figure 3). b. To place the slide(s) on the PALM stage (Figure 2B, C), click on button A (Load position) in the software (Figure 3A); the stage will move. Place slide(s) and click again on button A to let the stage return to the working position. c. To locate the tissue of interest, select the appropriate objective by clicking on button B in the software (Figure 3A) where an objective, with which the PALM is equipped, can be selected (5×, 10×, 20×, 40×, 63×). With the help of the correct objective, locate the tissues of interest. Use button C to help focus (Autofocus; Figure 3A). Note: In our work, depending on the gynoecium stage/size, we used either the 10× or the 20× objective. d. To select the tissue to be isolated by LAM, first select the tissue to isolate with the drawing tool in the software by clicking on button D (Freehand; Figure 3A). The drawing tool can be manipulated with the mouse or with the special pencil on the touch-sensitive monitor. Start selecting the tissue to isolate by drawing with the pen a closed circle/region of interest on the screen (Figure 3B, indicated with a red line); the laser will later cut over this drawn line. Next, to mark the catapulting of the selected tissue, click on button E (Dot; Figure 3A) and, with the pencil, mark by dots (or single dot) inside the previously drawn region to be catapulted (Figure 3B, indicated with blue dots), toward the collecting tubes (Figure 2C, E). After this process, reactivate the selection option by clicking on button F (Pointer; Figure 3A). Note: Many regions can be selected and collected by cutting and catapulting in series. In our work, per gynoecium, we collected the medial and lateral domains (in two different tubes); subsequently, we repeated it for the next gynoecium, and so on. In our hands, selecting regions in many gynoecia and then trying to collect all selected tissues did not work well, resulting, at a certain moment, in not properly cutting (not cutting completely through the section; probably loss of focus) or not accurately. e. To collect the selected tissue, place the TubeCollector 2×200 CM II on the microscope CapMover with the collecting tube(s) placed (Figure 2D, E). Click on button G (Cap Check; Figure 3A); the collecting tube will travel from its initial position to the position a few millimeters above the slide with the sample to be collected. To isolate and catapult the tissue of interest, activate the laser by clicking the button H (Figure 3A) and then let the sequence of cutting and catapulting actions run by clicking the button I (Start Cutting Laser; Figure 3B, Video 1). Notes: i. Before starting the process of cutting and catapulting the tissues of interest, test the laser energy Cite as: Luna-García, V. and de Folter, S. (2024). Laser-Assisted Microdissection and High-Throughput RNA Sequencing of the Arabidopsis Gynoecium Medial and Lateral Domains. Bio-protocol 14(17): e5056. DOI: 10.21769/BioProtoc.5056. 8 Published: Sep 05, 2024 and thickness to find the ideal working parameters by modifying the parameters of the part of the software indicated by the letter J (Energy and Focus; Figure 3A). Do this by trial and error prior to a real experiment. In our experiment, as a guideline, we used the following settings: laser cut energy 50–70; speed 10–50; LPC energy 60–75. Cut Focus and LPC focus change when the focus of the microscope changes, so we did not adjust them manually. The number of cycles of laser cutting is normally one. The z-focus parameter was not changed manually. ii. Use a single collection tube to collect a single tissue type of interest; you can perform this step as many times as necessary using the same tube to collect enough biological material for a good amount of RNA. iii. For our experiment, for one tissue type (for one RNA-seq sample), it took three days to collect the tissue from 20–30 slides with inflorescence tissue (i.e., different gynoecia stages were present, and only specific stages were selected for our experiment). iv. In general, microdissected material of around 80 gynoecia (sections of around 24 paraffin blocks with three or four embedded inflorescences) was pooled to result in one tissue sample (one replicate). Figure 2. PALM microscope. A. PALM microscope, with which the laser-microdissection of the tissues of interest is performed. B, C. Area of the stage, where the slide with the transverse sections (previously deparaffinized), as well as the TubeCollector 2×200 CM II, are placed. D, E. TubeCollector 2×200 CM II and AdhesiveCap 200 clear tubes. Cite as: Luna-García, V. and de Folter, S. (2024). Laser-Assisted Microdissection and High-Throughput RNA Sequencing of the Arabidopsis Gynoecium Medial and Lateral Domains. Bio-protocol 14(17): e5056. DOI: 10.21769/BioProtoc.5056. 9 Published: Sep 05, 2024 Video 1. Laser-assisted microdissection (LAM) of medial and lateral gynoecium tissues Figure 3. PALM software. A. PALM Robo v4 software and various parameters used for microdissection of tissues of interest. B. The area to be cut by the laser is in red and the amount of spot energy to be applied