This work was supported by the National Institute of Dental and Craniofacial Research (R01DE022988) and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (P01HD078233). The authors thank the Biorepository and Pathology Core for access to the Leica LMD 6500 platform at the Icahn School of Medicine at Mount Sinai.

Tissues from mice were obtained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and study protocols were approved by the Institutional Animal Care and Use Committee at the Icahn School of Medicine at Mount Sinai.
1. Preparation of Fresh Frozen Specimen
<ol>
	<li>Dissect the embryo or tissue of interest. Embed the sample in a disposable embedding mold with optimal cutting temperature (OCT) compound. Adjust the orientation of the specimen with a tip or needle.</li>
	<li>Rapidly freeze the samples in a dry ice/methyl-2-butane bath. Continue to the next step or store at -80 &#176;C. 
	NOTE: The protocol can be paused here.</li>
</ol>
2. Cryosectioning for Laser Capture Microdissection
<ol>
	<li>Defrost the cryostat and clean internal surfaces with 70% ethanol. Treat the anti-roll plate and a pair of forceps with RNase decontamination agent. Set the cryostat to the desired cutting temperature (-18 to -22 &#176;C). Prepare a lidded container with dry ice, placing a sheet of aluminum foil on the dry ice for temporary storage of the sectioned sample slides during cryosectioning. 
	CAUTION: Dry ice is extremely cold. Always handle dry ice with care and wear insulated gloves whenever handling it.</li>
	<li>Transfer the fresh frozen specimen into a bucket of dry ice. Place a layer of OCT compound onto a cryostat specimen holder and immediately place the specimen on top of the OCT with gentle pressure. When OCT is completely frozen, fasten the holder with the specimen into the cryostat cutting arm.</li>
	<li>Leave the sample in the cryostat for 15 min to equilibrate to the cutting temperature.</li>
	<li>Section the tissue and collect the slices on polyethylene naphthalate (PEN) membrane slides at a thickness of 12 &#956;m. Align consecutive sections on the membrane. Once one slide is completed, allow sections to dry for a few minutes and transfer the slide onto the foil in the dry ice container until all the required slides are collected.</li>
	<li>Store the slides at -80 &#176;C in a slide box. 
	NOTE:Section thickness is chosen to maximize the amount of tissue collected while allowing efficient cutting of the tissue, and may vary depending on tissue of interest. The protocol can be paused here. Keep slides free from RNase contamination. Avoid temperature changes. Although the RINs of RNA from slides stored for up to 6 months may still indicate high quality of the RNA, we recommend using slides as soon as possible.</li>
</ol>
3. Sample Preparation for Laser Capture Microdissection
<ol>
	<li>Prepare solutions for staining and dehydration.
	<ol>
		<li>Place 45 mL of 80% ethanol in each of three 50 mL centrifuge tubes on ice. Label them as #1, #2, and #3. Dilute ethanol with RNase/DNase free water.</li>
		<li>Place 45 mL of 95% ethanol in a 50 mL centrifuge tube on ice.</li>
		<li>Place 45 mL of 100% ethanol in a 50 mL centrifuge tube on ice.</li>
		<li>Place 45 mL of xylene in a 50 mL centrifuge tube at room temperature. 
		CAUTION: Xylene should be used in a fume hood.</li>
	</ol>
	</li>
	<li>Thaw a PEN membrane slide briefly by placing it against a gloved hand, then wash twice for 30 s each in 45 mL of 80% ethanol (#1 and #2) with agitation in 50 mL centrifuge tubes to remove the OCT. 
	NOTE: It is important to remove OCT before staining, to prevent OCT loosened during staining from obscuring the sections. Forceps can be used to facilitate removal of OCT that is not washed off by agitation.</li>
	<li>Lay slide on a sheet of aluminum foil. Pipette 0.8 mL of 0.1% cresyl violet in 50% ethanol onto the slide and stain for 30 s. Wash the slide in 45 mL of 80% ethanol (#3) for 30 s, and then dehydrate by passage for 30 s each through 45 mL of 95% ethanol, 100% ethanol, and xylene in 50 mL centrifuge tubes.</li>
	<li>Stand slides on a delicate task wiper for 5 min to drain xylene and dry sections. 
	NOTE: Slides must be dried completely before LCM.</li>
</ol>
4. Laser Capture Microdissection
NOTE: Wear powder free nitrile gloves while performing LCM.
<ol>
	<li>Turn on the LCM microscope, laser, and computer. Log on to computer and start the software. Turn the key to the "On" position to start up the laser. When the green light (Laser) is on, press the red button to activate the laser, and then the light (Laser) turns red indicating laser is ready to use. 
	NOTE: The Laser needs approximately 10 min to warm up.</li>
	<li>Set up the collection tubes.
	<ol>
		<li>Insert the cap of a 0.5 mL polymerase chain reaction (PCR) tube into the collector. 
		NOTE: Choose the matching collector for the desired PCR tubes (0.2 or 0.5 mL).</li>
		<li>Pipette 50 &#181;L of extraction buffer (provided by RNA isolation kit listed in the Table of Materials) into the cap of the 0.5 mL collection tube.</li>
		<li>Insert the collection device into the microscope.</li>
		<li>In the Change Collector Device window, click on the Move to Reference Point button to move the collector to the reference point (RP). Adjust the focus to clearly view the RP. Click the arrows to move the RP to the center of the view.</li>
	</ol>
	</li>
	<li>Load specimen slides.
	<ol>
		<li>Click the left Unload button in the toolbar to lower the slide holder.</li>
		<li>Place the slide into the holder, with the tissue section facing downwards. 
		NOTE: This orientation ensures that the captured tissue sections fall into the cap of the collection tube by gravity.</li>
		<li>Insert the holder back into the stage.</li>
	</ol>
	</li>
	<li>Set up the laser parameters.
	<ol>
		<li>Select the Control option of the Laser menu or click on the Laser icon.</li>
		<li>Adjust these parameters as desired: Power, the power of the laser; Aperture, the width of the laser; and Speed, the speed of cutting in Draw and Cut mode. 
		NOTE: Test the laser in an area out of interest. Higher power or larger aperture makes it easier to cut but causes more damage to cells. Adjust the combination of power, aperture, and speed to obtain efficient cutting and minimal damage of the tissue. For example, Power = 40, Aperture = 5, and Speed = 7 for cartilage tissue on a 12 &#956;m section.</li>
	</ol>
	</li>
	<li>Select and cut areas of interest.
	<ol>
		<li>Start with 5x objective and find the areas of interest, and then switch to the objective (5x, 10x, or 40x) that best shows the area of interest. 
		NOTE: After cresyl violet staining, cartilages are stained magenta and mineralized tissues appear brown or black, distinguishable from other tissues. Images can be saved using Save Image As from the File menu.</li>
		<li>Choose the tube for collection (A, B, C, D) by clicking the mark at the collector.</li>
		<li>Choose Move and Cut or Draw and Cut. In Move and Cut mode, the specimen is cut manually, using the mouse or the touch-screen pen to draw shapes freehand. In Draw and Cut mode, shapes may be drawn freehand with the mouse or touch-screen pen for subsequent cutting. Click the Start Cut button to initiate laser cutting.</li>
		<li>Once the cut is completed, the target tissue with PEN membrane falls into the cap of the collection tube by gravity. Repeat 4.5 to pool multiple areas of interest if needed. 
		NOTE: Repeating cuts or increasing Power or Aperture may be necessary if the tissue does not drop into the cap of the collection tube due to incomplete cutting. Mineralized bone (brown or black) cannot be cut directly by laser.</li>
	</ol>
	</li>
	<li>Unload the collector and carefully close the PCR tube. Place the microdissected tissues in dry ice. Continue to the next step or store at -80 &#176;C. 
	NOTE: The protocol can be paused here. Repeat 4.2 to 4.6 for the next sample.</li>
</ol>
5. Lysis of Microdissected Tissues and RNA Isolation
<ol>
	<li>Thaw the microdissected tissues at room temperature. Centrifuge briefly and incubate the samples for 30 min at 42 &#176;C.</li>
	<li>After incubation, spin the lysis buffer down into the 0.5 mL PCR tube. Perform DNase treatment and RNA extraction using an RNA isolation kit (see the Table of Materials) following the manufacturer's instructions.</li>
</ol>

The authors have nothing to disclose.

LCM enables the isolation of enriched or homogenous cell populations from heterogeneous tissues. Its advantages include rapid and precise capture of cells in their in vivo context, while potential disadvantages include it being time consuming, expensive, and limited by the need for the user to recognize distinct subpopulations within a specified sample30. This protocol provides details of LCM of mouse embryonic cartilage and bone, highlighting the use of cresyl violet staining in a rapid procedure to visualize cartilage and bone for precise tissue collection while maintaining high RNA integrity for subsequent analysis by RNA-seq. One limitation of this protocol is that the LCM system used here is not able to directly cut across mineralized tissues (e.g., the mandibular tissue in black in Figure 2C); as a result, the whole bone area needs to be dissected. Notably, the microdissected ossified regions isolated by LCM are not homogeneous, and include at least the subpopulations of osteoblasts, osteocytes, and osteoclasts (Table 1). Nevertheless, the enrichment by LCM is valuable as our previous study has shown that transcriptional changes in specific subpopulations such as osteoclasts in the microdissected bone tissue can still be detected by RNA-seq6.
For gene expression analysis, we have optimized the sample preparation and LCM procedure for high RNA quality and yield. Our protocol starts with fresh frozen tissues. Fresh frozen tissue sections allow for excellent quantity and quality of extracted RNA3, as mRNA is sensitive to standard methods of fixation31. RNA is quickly degraded by RNase contamination, and maintenance of an RNase-free environment throughout sample preparation, LCM, and RNA isolation is critical for successful application. Exposure to water during section processing is detrimental to RNA quality31,32. Our protocol limits such exposure, with the highest level of aqueous exposure being 50% during cresyl violet staining. We have tested that a quick staining (30 s) with 0.1% cresyl violet in 50% ethanol gives a distinguishable color to cartilage and does not lower the RNA integrity for downstream analysis such as low input RNA-seq (Figure 3C and Figure 4). The yield/area (ng/mm2) is approximately 20 ng/mm2 (Figure 3F), similar to or higher than previous optimized methods33. According to the cell densities in MC and mandibular bone6, we estimate that with this protocol the average yield from one cell is approximately 5 pg RNA per cell, and 1&ndash;5 ng of total RNA can be extracted from 200&ndash;1,000 cells, which can be used for low-input RNA-seq6,7,33. This yield efficiency is much higher than established LCM methods34, and also superior or similar to recent optimized protocols33,35.
The ability to distinguish different cell types in histological sections is essential for precise collection of specific tissues of interest. Cresyl violet is a hydrophilic, basic stain that binds to negatively charged nucleic acids36. This property makes it useful for counterstaining with cell-type selectivity37, and it is a standard histological stain for neurons38. Cresyl violet has been used in LCM for a variety of tissues, providing good tissue morphology and high RNA quality33,36,39,40. Compared with other staining methods, cresyl violet staining provides cytoplasmic and nuclear details, and a low RNA degradation rate32. We demonstrate here that cresyl violet staining is an easy and quick method to visualize cartilage and bone and that tissue stained with this method conserves high RNA integrity. Xylene treatment is commonly used for dehydration of the tissues before LCM35,36,41,42. We use xylene to enhance the visualization of tissue morphology35 which is essential to distinguish targeted tissues, especially on PEN membrane slides that are not as optically clear as plain glass slides. We found no significant occurrence of section loss from PEN slides during staining and washing steps, even with agitation to remove OCT.
Microdroplet or microfluidics-based single-cell RNA-seq (scRNA-seq) is a high-throughput method to analyze transcriptomes of tissues with heterogeneous cell types and has begun to be used in skeletal biology43. In contrast to LCM, scRNA-seq involves enzymatic and/or mechanical disaggregation of tissue into single cells. Most protocols for single cell preparation require tissues to be incubated at room temperature or 37 &deg;C for extended periods, which alters the transcriptome44,45. In addition, isolation of single cells in cartilage and bone may be physically limited by skeletal element complexity of trabecularization and mineralization. It is still a technical challenge to isolate a sufficient number of viable cells from skeletal tissues that are accurately representative of the cellular diversity of tissues in vivo, and incomplete dissociation of the cells may cause bias in the detection of cell types43. While scRNA-seq allows identification of distinct cell types, the sequencing depth is low, and typical sequencing approaches capture 3&rsquo; transcript ends and do not allow alternative splicing analysis. Bulk RNA-seq of LCM-derived tissue homogenizes cell differences, but allows comprehensive transcript detection and alternative splicing analysis. LCM is therefore especially useful for skeletal tissues that are not easily separable from surrounding tissues and internally complex, and fresh frozen preparation of the tissue can preserve both transcriptional profiles and RNA integrity for transcriptional analysis. Another weakness of scRNA-seq is that after single cell preparation, the spatial information of the cells is lost. A combination of LCM and scRNA-seq has been developed to permit the study of the transcriptome of a small sample from defined geographical locations (Geo-seq)46, which is another approach of utilizing LCM to study regionalized gene expression.
In summary, this protocol provides details of optimized LCM of cartilage and bone, highlighting the use of cresyl violet staining in a rapid procedure to visualize cartilage and bone for precise tissue collection while maintaining high RNA integrity for subsequent analysis by RNA-seq. This protocol has been used successfully for LCM of cartilage and bone at different developmental stages for gene expression analysis6,7, and also can be used for other tissues.

The musculoskeletal system is a multicomponent system composed of muscle, connective tissue, tendon, ligament, cartilage, and bone, innervated by nerves and vascularized by blood vessels1. The skeletal tissues develop with increasing cellular heterogeneity and structural complexity. Cartilage and bone develop from the same osteochondroprogenitor lineage and are highly related. Embryonic cartilage and bone develop in association with muscles, nerves, blood vessels, and undifferentiated mesenchyme. Cartilage may also be surrounded by bone, such as Meckel's cartilage and condylar cartilage within the mandibular bone. These tissues are anatomically associated and interact with each other through extracellular signals during development. In the study of gene expression in the development of cartilage and bone, one obstacle is the heterogeneity of skeletal structures composed of multiple tissue types. Precise isolation of the specific tissue of interest is key for successful transcriptional analysis.
Laser capture microdissection (LCM) is a powerful tool to isolate cell types or regions of interest within heterogeneous tissues, and is reproducible and is sensitive to the single cell level2. It can precisely target and capture cells of interest for a wide range of downstream assays in transcriptomics, genomics, and proteomics3,4. The quality of the isolated RNA, DNA, or protein can be assessed with a bioanalyzer or equivalent platform. For example, RNA quality is indicated by the RNA integrity number (RIN)5.
Here, we provide a protocol for the rapid staining and isolation of cartilage and bone by LCM from fresh frozen tissues. We use the mouse embryo to demonstrate that this protocol yields high quality RNA for subsequent transcriptomic analysis, such as RNA sequencing (RNA-seq).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Methylbutane</td>
			<td>ThermoFisher Scientific</td>
			<td>O3551-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioanalyzer</td>
			<td>Agilent</td>
			<td>G2939BA</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>ThermoFisher Scientific</td>
			<td>339653</td>
			<td>Conical sterile polypropylene centrifuge tubes, 50 mL</td>
		</tr>
		<tr>
			<td>Cresyl violet acetate</td>
			<td>Sigma-Aldrich</td>
			<td>C5042</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica Biosystems</td>
			<td>CM3050 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Delicate task wiper</td>
			<td>ThermoFisher Scientific</td>
			<td>06-666</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable embedding mold</td>
			<td>ThermoFisher Scientific</td>
			<td>1220</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td>Invitrogen</td>
			<td>10977-015</td>
			<td>DNase/RNase-Free</td>
		</tr>
		<tr>
			<td>Ethanol, absolute (200 proof)</td>
			<td>ThermoFisher Scientific</td>
			<td>BP2818</td>
			<td>Molecular biology grade</td>
		</tr>
		<tr>
			<td>Glass PEN membrane slide</td>
			<td>Leica Microsystems</td>
			<td>11505158</td>
			<td></td>
		</tr>
		<tr>
			<td>LCM system</td>
			<td>Leica Microsystems</td>
			<td>Leica LMD6500</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass</td>
			<td>ThermoFisher Scientific</td>
			<td>12-545FP</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>ThermoFisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT compound</td>
			<td>Electron Microscopy Sciences</td>
			<td>102094-106</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tube with flat cap, 0.5 mL</td>
			<td>Axygen</td>
			<td>PCR-05-C</td>
			<td>LCM collection tubes</td>
		</tr>
		<tr>
			<td>Permanent mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA isolation kit</td>
			<td>ThermoFisher Scientific</td>
			<td>KIT0204</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase decontamination agent</td>
			<td>Sigma-Aldrich</td>
			<td>R2020</td>
			<td>RNase decontamination agent for cleaning surfaces</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma-Aldrich</td>
			<td>214736</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser capture microdissection of mouse embryonic cartilage and bone for gene expression analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

Coronal sections of fresh frozen mouse tissues at E16.5 were used to demonstrate the isolation and collection of Meckel&#39;s cartilage (MC), condylar cartilage, and mandibular bone by LCM. Mouse embryos at E16.5 were dissected and embedded in cryogenic molds with OCT compound. Samples in molds were rapidly frozen in a dry ice and methyl-2-butane bath and stored at -80 &#176;C.
To demonstrate cresyl violet staining of cartilage and bone, cryosectioning in the coronal plane was performed and samples were collected on microscope slides. Sections were washed, stained, and dehydrated following the protocol above (step 3.2&#8211;3.4). Slides were air dried and mounted with permanent mounting medium. All cartilages examined were stained magenta (MC, condylar cartilage, nasal septum cartilage, costal cartilage, cartilage primordium of presphenoid bone, cartilage primordia of radius and ulna) and all mineralized tissues were stained brown or black (Figure 1A&#8211;E). Both cartilage and bone were easily distinguished from other tissues at multiple anatomical sites.
For LCM, heads of embryos at E16.5 were sectioned in the coronal plane on PEN membrane slides at a thickness of 12 &#956;m, and 6&#8211;8 consecutive sections were collected per slide and stored at -80 &#176;C. OCT was removed and sections were stained with cresyl violet and dehydrated following the protocol described above (step 3.2&#8211;3.4). MC, condylar cartilage, and the mandibular bone regions were selected and isolated by LCM (Figure 2). The selected regions dropped into the lysis buffer in the cap of the collection tube. To obtain sufficient RNA for sequencing, we pooled 10 regions of MC, 10 regions of condylar cartilage or 4 regions of mandibular bone into each collection tube as one sample, respectively.
RNA was extracted using an RNA isolation kit following the manufacturer&#39;s instructions. Total RNA was analyzed using a bioanalyzer (Figure 3A&#8211;B). To test the effect of cresyl violet staining on the quality of RNA, we compared RNA from mandibular bone samples stained with cresyl violet and samples without staining (mineralized tissue is visible without staining, but cresyl violet staining enhances the visibility to distinguish bone from surrounding tissues). No significant difference in RNA integrity was observed between stained samples (n = 4) and samples without staining (n = 4), indicating the quick cresyl violet staining in this protocol has insignificant effect on RNA quality (P = 0.858, two-tailed Welch&#39;s t-test, Figure 3C). We used our protocol for LCM of various tissues at different developmental stages and RINs were measured, indicating high RNA quality (Figure 3D). Average yields of RNA from MC, condylar cartilage, and mandibular bone were 7.50 &#177; 1.45 ng, 12.55 &#177; 2.75 ng, and 33.02 &#177; 7.63 ng (Figure 3E) and the yield/area was 19.73 &#177; 3.82 ng/mm2, 26.70 &#177; 5.84 ng/mm2, and 17.23 &#177; 3.98 ng/mm2, respectively (Figure 3F), without significant difference among tissues (MC versus condylar cartilage, P = 0.383; condylar cartilage versus mandibular bone, P = 0.260; MC versus mandibular bone, P = 0.674).
Libraries were prepared and sequenced as previously described6,7. A representative cDNA size was approximately 500 bp (Figure 4A). RNA-seq data was analyzed with MultiQC8. We analyzed RNA-seq data from 18 LCM samples (MC1-6, Meckel&#39;s cartilage; C1-6, condylar cartilage; M1-6, mandibular bone). The mean quality values across each base position in the reads were generated by FastQC, indicating very good quality calls (Figure 4B). Read alignment was analyzed with Picard (Figure 4C). The reads showed high aligned percentages and the average percentage of aligned reads was 75%. Gene coverage was analyzed with Picard (Figure 4D), which indicated a good representation along the transcript from the 5&#39; to the 3&#39; end.
Differential gene expression analysis was performed6,7. There were 4,006 genes significantly differentially expressed (P &#60; 0.05) between the mandibular bone and MC (Figure 5A). Genes specific to osteoblasts or osteocytes (Col1a19, Col1a210, Dkk111, Dmp112, Dstn13, Runx214, Sp715, and Sparc16) were more highly expressed in the mandibular bone compared to MC, while chondrocyte-specific genes (Acan17, Col2a118, Col9a119, Col9a220, Col9a321, Comp22, Lect123, and Sox524) were more highly expressed in MC compared to the mandibular bone (Figure 5B and Table 1). In addition, osteoclast markers such as Acp525, Csf1r26, Ctsk27, Itgb328, and Oscar29 were also identified as more highly expressed in the mandibular bone compared to MC (Figure 5B and Table 1), indicating successful isolation of targeted tissues.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60503/60503fig1a.jpg" /> 
Figure 1: Representative cresyl violet staining of cartilage and bone in coronal sections of the mouse embryo at E16.5. (A) Meckel&#39;s cartilage and hemimandible. (B) Meckel&#39;s cartilage, condylar cartilage, and hemimandible. (C) Nasal septum and maxillae. (D) Cartilage primordium of presphenoid bone and palatine bone. (E) Cartilage primordium of radius, cartilage primordium of ulna, and costal cartilage. C = condylar cartilage; CC = costal cartilage; CP = cartilage primordium of presphenoid bone; M = hemimandible; MC = Meckel&#39;s cartilage; MX = maxilla; NS = nasal septum; PL = palatine bone; R = cartilage primordium of radius; U = cartilage primordium of ulna. Scale bar = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60503/60503fig1alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60503/60503fig02.jpg" /> 
Figure 2: Representative regions isolated by LCM and collected for RNA-seq. (A&#8211;C) Representative stained MC (A), condylar cartilage (B), and hemimandible with MC already isolated (C) in cryosection before LCM. (D&#8211;F) The regions in A, B, and C after targeted tissues were isolated by LCM. Scale bar = 200 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60503/60503fig2blarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60503/60503fig3b.jpg" /> 
Figure 3: RNA quality and quantity of LCM samples. (A&#8211;B) Representative electropherogram (A) and the associated gel image (B) from a bioanalyzer for a mandibular bone sample. (C) RINs of total RNA from mandibular bone samples stained with cresyl violet (n = 4) or without staining (n = 4). (D) RINs of total RNA from different tissues isolated by LCM. Meckel&#39;s cartilage at E16.5 (n = 6); Condylar cartilage at E16.5 (n = 6); Mandibular bone at E16.5 (n = 6); Nasal septum cartilage at E14.5 (n = 6); Brain at E14.5 (n = 6); Brain at E16.5 (n = 7); Brain at E18.5 (n = 4). (E) Yield of total RNA from three tissues. Each MC or condylar cartilage sample was a pool of 10 microdissected regions of cartilage and each sample of mandibular bone was a pool of 4 microdissected regions of hemimandible. (F) The yield per unit area (ng/mm2) in each tissue. Data are mean &#177; s.e.m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60503/60503fig3blarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60503/60503fig4a.jpg" /> 
Figure 4: The quality of libraries and RNA-seq data generated from LCM samples. (A) Representative cDNA sizes of a library from a mandibular bone sample determined by a bioanalyzer. (B&#8211;D) Quality control analysis of RNA-seq from 18 LCM samples (MC1-6, Meckel&#39;s cartilage; C1-6, condylar cartilage; M1-6, mandibular bone) by MultiQC. (B) The mean quality values across each base position in the reads were generated by FastQC. The background of the graph divides the y axis into very good quality calls (green), calls of reasonable quality (orange), and calls of poor quality (red). (C) Alignment of reads was analyzed by Picard. The summary is shown as the percentages of aligned reads. (D) Normalized gene coverage analyzed with Picard. The plot indicates the relative average coverage along the transcript from 5&#39; end (left) to 3&#39; end (right). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60503/60503fig4alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60503/60503fig05.jpg" /> 
Figure 5: Differential expression analysis of RNA-seq data from mandibular bone and MC isolated by LCM. (A) Hierarchical clustering of 4,006 genes significantly differentially expressed (P &#60; 0.05) between the mandibular bone and MC. Three biological replicates were used for each tissue. M1-3, mandibular bone; MC1-3, MC. (B) Volcano plot showing fold changes and P-values of differentially expressed genes between mandibular bone and MC. Examples of highly differentially expressed cell-specific genes are shown: osteoblast and osteocyte markers in blue, osteoclast markers in green, and chondrocyte markers in red. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60503/60503fig5blarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Gene</td>
			<td>Cell type</td>
			<td>log2FoldChange</td>
			<td>Average Expression</td>
			<td>Adjusted P Value</td>
		</tr>
		<tr>
			<td>Col1a1</td>
			<td>Osteoblast/Osteocyte</td>
			<td>2.64</td>
			<td>12.94</td>
			<td>2.22E-05</td>
		</tr>
		<tr>
			<td>Col1a2</td>
			<td>Osteoblast/Osteocyte</td>
			<td>3.41</td>
			<td>12.87</td>
			<td>8.27E-07</td>
		</tr>
		<tr>
			<td>Dkk1</td>
			<td>Osteoblast/Osteocyte</td>
			<td>7.59</td>
			<td>2.01</td>
			<td>5.21E-03</td>
		</tr>
		<tr>
			<td>Dmp1</td>
			<td>Osteoblast/Osteocyte</td>
			<td>9.73</td>
			<td>3.42</td>
			<td>3.19E-04</td>
		</tr>
		<tr>
			<td>Dstn</td>
			<td>Osteoblast/Osteocyte</td>
			<td>2.51</td>
			<td>6.87</td>
			<td>5.73E-07</td>
		</tr>
		<tr>
			<td>Runx2</td>
			<td>Osteoblast/Osteocyte</td>
			<td>1.24</td>
			<td>8.62</td>
			<td>4.08E-02</td>
		</tr>
		<tr>
			<td>Sp7</td>
			<td>Osteoblast/Osteocyte</td>
			<td>2.24</td>
			<td>6.95</td>
			<td>5.81E-03</td>
		</tr>
		<tr>
			<td>Sparc</td>
			<td>Osteoblast/Osteocyte</td>
			<td>3.40</td>
			<td>12.26</td>
			<td>5.56E-09</td>
		</tr>
		<tr>
			<td>Acp5</td>
			<td>Osteoclast</td>
			<td>3.38</td>
			<td>5.74</td>
			<td>6.46E-06</td>
		</tr>
		<tr>
			<td>Csf1r</td>
			<td>Osteoclast</td>
			<td>2.01</td>
			<td>5.80</td>
			<td>1.17E-04</td>
		</tr>
		<tr>
			<td>Ctsk</td>
			<td>Osteoclast</td>
			<td>3.19</td>
			<td>7.56</td>
			<td>5.73E-07</td>
		</tr>
		<tr>
			<td>Itgb3</td>
			<td>Osteoclast</td>
			<td>2.88</td>
			<td>5.37</td>
			<td>2.43E-04</td>
		</tr>
		<tr>
			<td>Oscar</td>
			<td>Osteoclast</td>
			<td>3.41</td>
			<td>2.67</td>
			<td>1.63E-04</td>
		</tr>
		<tr>
			<td>Acan</td>
			<td>Chondrocyte</td>
			<td>-5.23</td>
			<td>5.01</td>
			<td>2.76E-04</td>
		</tr>
		<tr>
			<td>Col2a1</td>
			<td>Chondrocyte</td>
			<td>-3.61</td>
			<td>9.47</td>
			<td>3.29E-03</td>
		</tr>
		<tr>
			<td>Col9a1</td>
			<td>Chondrocyte</td>
			<td>-7.01</td>
			<td>3.58</td>
			<td>5.51E-03</td>
		</tr>
		<tr>
			<td>Col9a2</td>
			<td>Chondrocyte</td>
			<td>-5.40</td>
			<td>3.76</td>
			<td>2.60E-03</td>
		</tr>
		<tr>
			<td>Col9a3</td>
			<td>Chondrocyte</td>
			<td>-6.63</td>
			<td>3.80</td>
			<td>2.90E-02</td>
		</tr>
		<tr>
			<td>Comp</td>
			<td>Chondrocyte</td>
			<td>-5.86</td>
			<td>1.54</td>
			<td>7.51E-03</td>
		</tr>
		<tr>
			<td>Lect1</td>
			<td>Chondrocyte</td>
			<td>-7.37</td>
			<td>1.34</td>
			<td>2.35E-02</td>
		</tr>
		<tr>
			<td>Sox5</td>
			<td>Chondrocyte</td>
			<td>-2.88</td>
			<td>4.43</td>
			<td>6.06E-04</td>
		</tr>
	</tbody>
</table>
Table 1: Differential expression of known genes in various cell types of bone and cartilage (mandibular bone versus MC).

Watch this Scientific Journal Video about Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis at JoVE.com

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

laser-capture-microdissection-mouse-embryonic-cartilage-bone-for-gene

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

7.0K Views.  Icahn School of Medicine at Mount Sinai. This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Video: Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, &#946;-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for &#946;-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the method for paraffin sectioning and counterstaining. This is an easy and quick procedure to monitor gene expression during development that can also be applied to tissue sections, organs or cultured cells.

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.