This work was supported by the following grants: F31-DK113666 (AWW), K01-DK102868 (AMZ), K08-DK106478 (KJW), and P30-DK050306 (Pilot grant to KJW).

All methods that involve the use of mice are consistent with the guidelines provided by the IACUC of the Penn Office of Animal Welfare at the University of Pennsylvania.
1. Reagent Preparation
<ol>
	<li>Cycloheximide
	<ol>
		<li>To make 500 &#181;L of 0.1 g/mL cycloheximide, suspend 50 mg of cycloheximide in 500 &#181;L of methanol. 
		CAUTION: Cycloheximide is extremely toxic to the environment and can cause congenital malformation. All wastes and buffers containing cycloheximide should be collected for proper disposal. 
		NOTE: Cycloheximide can be stored at 4 &#176;C for up to 1 day. Cycloheximide inhibits translation.</li>
	</ol>
	</li>
	<li>Dithiothreitol (DTT)
	<ol>
		<li>To make 1 mL of 1 M DTT, suspend 0.15 g of DTT powder in RNase-free water. 
		CAUTION: DTT can cause irritation to the skin, eye, and respiratory tract. 
		NOTE: DTT is a detergent. DTT can be stored at -20 &#176;C. It is recommended to store 1 M DTT in single-use aliquots of 50 &#181;L.</li>
	</ol>
	</li>
	<li>Deoxycholate (DOC)
	<ol>
		<li>To make 10% DOC, suspend 1 g of DOC in a 50 mL conical tube and add RNase-free water up to 10 mL. Shake vigorously until the powder is dissolved. 
		NOTE: The 10% DOC solution is slightly yellow and can be stored at room temperature (RT) for up to 1 year. DOC is used for nuclear lysis.</li>
	</ol>
	</li>
	<li>GFP antibodies
	<ol>
		<li>Aliquot GFP antibodies when using for the first time. Snap freeze the aliquots and store at -80 &#176;C. 
		NOTE: It is recommended to store 50 &#181;g of GFP antibodies in single-use aliquots.</li>
	</ol>
	</li>
	<li>B biotinylated protein L
	<ol>
		<li>Resuspend biotinylated protein L in 1x phosphate-buffered saline (PBS) to make the final concentration 1 &#181;g/&#181;L. 
		NOTE: Resuspended solution can be stored at -20 &#176;C for up to 6 months.</li>
	</ol>
	</li>
</ol>
2. Buffer Preparation
<ol>
	<li>Bovine serum albumin (BSA) buffer
	<ol>
		<li>To make 50 mL of 3% BSA buffer, add 1.5 g of IgG- and protease-free BSA powder into 40 mL of PBS followed by vortex. After the BSA is dissolved, add PBS to a final volume of 50 mL. 
		NOTE: The BSA buffer can be stored at 4 &#176;C for up to six months.</li>
	</ol>
	</li>
	<li>Dissection buffer
	<ol>
		<li>To make 50 mL of dissection buffer stock, combine 5 mL of 10x Hank&#39;s balanced salt solution (HBSS), 125 &#181;L of 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1,750 &#181;L of 1 M glucose, and 200 &#181;L of 1 M NaHCO3. Add RNase-free water to a final volume of 50 mL. 
		NOTE: The dissection buffer stock can be stored at 4 &#176;C for up to six months.</li>
		<li>Immediately prior to use, add 100 &#181;g/mL of 0.1 g/mL cycloheximide and keep on ice.</li>
	</ol>
	</li>
	<li>High-salt buffer
	<ol>
		<li>To make 50 mL of high-salt buffer stock, add 1 mL of 1 M HEPES, 8.75 mL of 2 M KCl, 500 &#181;L of 1 M MgCl2, and 500 &#181;L of 100% nonylphenyl polyethylene glycol (Table of Materials) to RNase-free water. 
		NOTE: The high-salt buffer stock can be stored at 4 &#176;C for up to six months.</li>
		<li>Immediately prior to use, add 0.5 &#181;L/mL of 1 M DTT and 1 &#181;L/mL of 0.1 g/mL cycloheximide. Keep the fresh high-salt buffer on ice.</li>
	</ol>
	</li>
	<li>Low-salt buffer
	<ol>
		<li>To make 50 mL of low-salt buffer stock, add 1 mL of 1 M HEPES, 3.75 mL of 2 M KCl, 500 &#181;L of 1 M MgCl2, and 500 &#181;L of 100% nonylphenyl polyethylene glycol to 44.25 Ml of RNase-free water. 
		NOTE: The low-salt buffer stock can be stored at 4 &#176;C for up to six months.</li>
		<li>Add 0.5 &#181;L/mL of 1 M DTT and 1 &#181;L/mL of 0.1 g/mL cycloheximide prior to use. Keep the fresh low-salt buffer on ice.</li>
	</ol>
	</li>
	<li>Tissue lysis buffer
	<ol>
		<li>To make 50 mL of tissue lysis buffer stock, combine 1 mL of 1 M HEPES, 3.75 mL of 2 M KCl, and 500 &#181;L of 1 M MgCl2. Add RNase-free water to a final of 50 mL. 
		NOTE: The dissection buffer stock can be stored at 4 &#176;C for up to six months.</li>
		<li>Add 1 tablet/10 mL of EDTA-free protease inhibitor, 1 &#181;L/mL of 0.1 g/mL cycloheximide, 10 &#181;L/mL of RNase inhibitors each immediately prior to use. Keep the fresh tissue lysis buffer on ice.</li>
	</ol>
	</li>
</ol>
3. Conjugation of Antibodies to Magnetic Beads
<ol>
	<li>Antibodies
	<ol>
		<li>Calculate the amount of GFP antibodies required for all samples and prepare for one extra sample. 
		NOTE: For each sample, 50 &#181;g of each GFP antibody is required.</li>
		<li>Thaw GFP antibodies on ice and spin at maximum speed (&#62;13,000 x g) for 10 min at 4 &#176;C and transfer supernatants to a new microcentrifuge tube. 
		NOTE: The antibody preparation step can be performed prior to bead preparation and the thawed antibodies can be kept on ice. Alternatively, this part can be performed during incubation of the magnetic bead and biotinylated protein L.</li>
	</ol>
	</li>
	<li>Resuspend magnetic beads
	<ol>
		<li>Resuspend magnetic beads (Table of Materials) by gentle pipetting.</li>
		<li>For each sample, use 150 &#181;L of magnetic bead. Calculate the volume of magnetic bead required for all samples and prepare one extra. Transfer the resuspended magnetic beads to a 1.5 mL or 2 mL microcentrifuge tube. If more than 1 mL is required for an experiment, split the total amount into equal volumes.</li>
		<li>Collect beads on a magnetic stand for &#62;1 min and remove the supernatant. Remove the microcentrifuge tube from the magnetic stand and add 1 mL of PBS followed by pipetting up and down to wash the beads. Collect beads on a magnetic stand for &#62;1 min and remove PBS.</li>
	</ol>
	</li>
	<li>Preparation of protein L-coated beads
	<ol>
		<li>For each sample, use 60 &#181;L of biotinylated protein L. If protein L is previously resuspended and stored at -20 &#176;C, thaw protein L on ice. If protein L is resuspended prior to use, take the amount required for all samples and prepare one extra.</li>
		<li>Add the calculated volume of biotinylated protein L to the resuspended and washed magnetic beads. Add 1x PBS to make the final volume 1 mL if using a 1.5 mL microcentrifuge tube, or 1.5 mL if using a 2 mL microcentrifuge tube. Incubate magnetic beads with biotinylated protein L for 35 min at RT on a tube rotator. 
		NOTE: Antibodies can be prepared at this step while the beads are incubating with biotinylated protein L.</li>
		<li>Collect protein L-coated beads on a magnetic stand for &#62;1 min and remove the supernatant. Remove the microcentrifuge tube from the magnetic stand and add 1 mL of 3% BSA buffer followed by gentle pipetting for at least 5 times to wash the protein L-coated beads.</li>
		<li>Collect coated beads on a magnetic stand for &#62;1 min and remove the supernatant. Repeat the washing steps with 3% BSA for another 4 times (a total of 5 times).</li>
	</ol>
	</li>
	<li>Antibody binding
	<ol>
		<li>Add the calculated amount of GFP antibodies into the protein L-coated beads and incubate for 1 h at 4 &#176;C on a tube rotator. 
		NOTE: After antibody incubation, take special care to not vortex the affinity matrix.</li>
		<li>During incubation, prepare low-salt buffer by calculating the total volume required for all samples and add 0.5 &#181;L/mL of 1 M DTT and 1 &#181;L/mL of 0.1 g/mL cycloheximide to low-salt buffer stock prior to use. 
		NOTE: 3 mL of low-salt buffer for washing each tube of GFP-conjugated beads and 200 &#181;L/sample for resuspension of the GFP-conjugated beads is required. Fresh low-salt buffer can be kept on ice for a couple of hours.</li>
		<li>Collect the affinity matrix on a magnetic stand for &#62;1 min and remove the supernatant. Add 1 mL of low-salt buffer and gently pipette up and down to wash the affinity matrix. Collect the affinity matrix on a magnetic stand for &#62;1 min and remove low-salt buffer. Repeat the washing steps with low-salt buffer for another 2 times (a total of 3 times).</li>
		<li>Resuspend the beads in low-salt buffer so that each sample has 200 &#181;L of affinity matrix. 
		NOTE: The affinity matrix can be stored in 0.02% NaN3 at 4 &#176;C for up to 2 weeks. The affinity matrix should be quickly washed in low-salt buffer 3 times and resuspended gently on a tube rotator at 4 &#176;C for at least 10 min if the affinity matrix is prepared within 1 week or overnight if the affinity matrix is stored for over 1 week. The protocol can be paused after this step. 
		CAUTION: Sodium azide is extremely toxic to the environment. Contact with acids produces toxic gas. All wastes should be collected for proper disposal.</li>
	</ol>
	</li>
</ol>
4. Liver Tissue Lysis
<ol>
	<li>Buffer preparation and equipment setup
	<ol>
		<li>Calculate the number of microcentrifuge tubes required, label and chill on ice. 
		NOTE: Usually, seven 1.5 mL microcentrifuge tubes are required for each sample: 1 for the remaining dissected liver, 4 for 4 mL of homogenized liver lysate, and 2 for transferring supernatants.</li>
		<li>Prepare fresh dissection buffer by calculating the total volume required for all samples and add 1 &#181;L/mL of 0.1 g/mL cycloheximide. Place the fresh dissection buffer on ice to keep cold throughout the experiment. 
		NOTE: For each sample, 10 mL of dissection buffer is required.</li>
		<li>Prepare fresh lysis buffer by calculating the total volume required for all samples and add 1 tablet/10 mL of EDTA-free protease inhibitor, 1 &#181;L/mL of 0.1 g/mL cycloheximide, and 10 &#181;L/mL of RNase inhibitors each. Keep the lysis buffer on ice throughout the experiment. 
		NOTE: For each sample, 4 mL of lysis buffer is required.</li>
		<li>Set up the tissue grinder (Table of Materials) so that the polytetrafluoroethylene (PTFE)-glass tubes can be placed on ice during homogenization of liver pieces. Put 4 mL of cold lysis buffer in the PTFE-glass tubes.</li>
	</ol>
	</li>
	<li>Repopulating liver homogenization
	<ol>
		<li>Euthanize an 8&#8722;12-week-old Fah-/- mouse injected with the TRAP vector and repopulated for 1&#8722;4 weeks with anesthesia and cervical dislocation according to approved animal experimental guidelines.</li>
		<li>Place the mouse on a dissection board and spray the abdomen with 70% ethanol. Tent the skin and peritoneum low in the abdomen using forceps and use scissors to make a transverse incision. Continue to cut with the scissors to make a wide U-shaped peritoneal flap, with care to not cut the viscera. Flip the peritoneal flap over the sternum to expose the liver.</li>
		<li>Carefully remove the liver using fine scissors and forceps and quickly place the tissue in cold dissection buffer to rinse. To homogenize frozen tissues, quickly move the desired amount of liver tissue into PTFE-glass tubes with cold lysis buffer without thawing the tissue. 
		NOTE: The dissected tissue can be flash-frozen and stored at -80 &#176;C after it is washed with dissection buffer. The protocol can be paused after this step.</li>
		<li>Weigh the liver on a Petri dish and Isolate 200&#8722;500 mg of liver pieces and move into the PTFE-glass tubes. Place the remaining liver tissue into a pre-chilled microcentrifuge tube and flash freeze.</li>
		<li>Homogenize the samples in a motor-driven homogenizer starting at 300 rpm to dissociate hepatocytes from the liver structure for at least 5 strokes. Lower the glass tube each time but take care to not let the pestle rise above the solution to prevent aeration that could cause protein denaturation.</li>
		<li>Raise the speed to 900 rpm to fully homogenize the liver tissues for at least 12 full strokes.</li>
		<li>Transfer the lysate into the labeled and pre-chilled tubes, with no more than 1 mL of lysate per 1.5 mL microcentrifuge tubes. If 4 mL of lysis buffer is used, keep 1 tube and flash freeze the remaining 3 tubes. 
		NOTE: The lysates can be kept on ice for up to 1 h while dissecting the next animal and preparing fresh lysates. The homogenized liver can be flash frozen after the lysis step and stored at -80 &#176;C. There could be a 50% decrease in isolated RNA if frozen lysates are used. The protocol can be paused after this step.</li>
	</ol>
	</li>
	<li>Nuclear lysis
	<ol>
		<li>Centrifuge the liver lysate at 2,000 x g at 4 &#176;C for 10 min and transfer the supernatant to a new, prechilled microcentrifuge tube on ice.</li>
		<li>Add 1/9 of the supernatant volume of 10% nonylphenyl polyethylene glycol to make the final concentration 1% nonylphenyl polyethylene glycol and mix by gently inverting the microcentrifuge tubes.</li>
		<li>Quickly spin down the microcentrifuge tubes and add 1/9 of the sample volume of 10% DOC to make the final concentration 1% DOC and mix by gently inverting the microcentrifuge tubes. Quickly spin down the microcentrifuge tubes and incubate on ice for 5 min.</li>
		<li>Centrifuge the nuclear lysate at 20,000 x g at 4 &#176;C for 10 min and transfer the supernatant to a new, prechilled microcentrifuge tube on ice. 
		NOTE: The mitochondria-depleted supernatant can be placed on ice for a couple of hours while the remaining samples are being collected.</li>
	</ol>
	</li>
</ol>
5. Immunoprecipitation
<ol>
	<li>For each tube, take out 1% of the total volume of the supernatant as a pre-immunoprecipitation control to compare target enrichment after incubation with the affinity matrix. Place pre-immunoprecipitation controls on a tube rotator at 4 &#176;C overnight, the same way as the immunoprecipitated samples are processed.</li>
	<li>Add 200 &#181;L of affinity matrix to each sample. Take extra care to resuspend the beads by gentle pipetting prior to adding the affinity matrix to each sample. Incubate the lysates with affinity matrix at 4 &#176;C overnight with gentle mixing on a tube rotator. 
	NOTE: The protocol can be paused for up to a day after this step.</li>
</ol>
6. RNA Isolation
<ol>
	<li>Buffer preparation and equipment setup
	<ol>
		<li>Place the magnetic rack at 4 &#176;C for at least 30 min to pre-chill and keep the rack on ice throughout the experiment.</li>
		<li>Calculate the number of microcentrifuge tubes required and pre-chill on ice or at 4 &#176;C. 
		NOTE: Usually, each sample requires 1 microcentrifuge tube for the final purified RNA.</li>
		<li>Quickly spin down the tubes from step 5.2 and collect the beads by placing on the magnetic rack for at least 1 min. Collect or discard the supernatant in additional microcentrifuge tubes. 
		NOTE: The collected supernatant that contains unbound fraction can be flash-frozen and stored at -80 &#176;C to compare with the bound fraction for transcript enrichment after purification. The protocol can be paused after this step.</li>
		<li>Prepare high-salt buffer by adding 0.5 &#181;L/mL of 1 M DTT and 1 &#181;L/mL of 0.1 g/mL cycloheximide to high-salt buffer stock. 
		NOTE: For each sample, 5 mL of high-salt buffer is required.</li>
	</ol>
	</li>
	<li>RNA isolation
	<ol>
		<li>Add 1 mL of fresh high-salt buffer to each tube followed by gentle pipetting for at least 5 times without introducing bubbles. 
		NOTE: Insufficient washing could introduce backgrounds of unbound transcripts while the introduction of bubbles could accelerate RNA degradation.</li>
		<li>Collect beads on a magnetic stand for &#62;1 min and remove the supernatant. Repeat the washing steps with high-salt buffer for another 4 times (a total of 5 times).</li>
		<li>Remove remaining high-salt buffer and remove microcentrifuge tubes from the magnetic stand and place at RT for 5 min to warm up.</li>
		<li>Resuspend the beads in 100 &#181;L of lysis buffer (provided in the RNA isolation kit) with &#946;-mercaptoethanol. 
		NOTE: Any RNA isolation and purification kit that contains the denaturant guanidine thiocyanate can be used as a lysis buffer to release bound RNA from the affinity matrix. RNA extraction should be processed at RT since guanidine thiocyanate can crystallize at low temperatures.</li>
		<li>Vortex the beads and buffer for at least 5 s at the highest speed, quickly spin down to collect the buffer on the side of the microcentrifuge tube and incubate the beads with the buffer at RT for 10 min to release the bead-bound RNA into the lysis buffer.</li>
		<li>Collect beads on a magnetic stand for &#62;1 min and collect the supernatant to proceed immediately to RNA cleanup according to the RNA purification protocol as specified in the kit (Table of Materials). 
		NOTE: The supernatant containing the eluted RNA in lysis buffer can also be stored at -80 &#176;C for up to 1 month before cleanup by warming up to RT upon thawing.</li>
		<li>To achieve maximum quality of the isolated RNA, perform all optional steps including DNase digestion and all RNA elution steps. Heat up the elution buffer provided by the RNA isolation kit or RNase-free water to 60 &#176;C for maximum RNA recovery. 
		NOTE: The isolated RNA can be stored at -20 &#176;C for up to 1 month or -80 &#176;C for several years. The protocol can be paused after this step.</li>
	</ol>
	</li>
</ol>
7. Optional RNA Quality Analysis (Recommended)
<ol>
	<li>Assess RNA quality using a bioanalyzer11 and quantity with a spectrophotometer12 to determine if repeating the immunoprecipitation process is required to obtain ample and high-quality RNA. 
	NOTE: The optimal RNA quality for high-throughput sequencing should follow protocols specified by library preparation kits and sequencing platforms.</li>
</ol>
8. Downstream Applications
NOTE: Total RNA isolated by the TRAP protocol can be used in a number of standard downstream applications, including RNA-seq (TRAP-seq). Standard reverse transcription and quantitative PCR protocols can also be used following TRAP.
<ol>
	<li>For TRAP-seq, perform RNA-seq library preparation according to standard methods13.</li>
	<li>For RNA-seq, prepare cDNA sequencing libraries using commercial RNA-seq kits with oligo d(T)-based enrichment of polyadenylated poly(A) transcripts. Alternatively, if the total RNA quality is lower than recommended for poly(A) enrichment, use rRNA depletion modules. However, expect to see more rRNA alignment after sequencing.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-Cell+RNA-Seq+of+the+Pancreatic+Islets--a+Promise+Not+yet+Fulfilled?.">Single-Cell RNA-Seq of the Pancreatic Islets--a Promise Not yet Fulfilled?.</a> Cell Metabolism. 29 (3), 539-544 (2019).</li></ol>

The authors have nothing to disclose.

TRAP-seq is a technique for cell type-specific isolation of translating mRNA via epitope-tagged ribosomes and presents an alternative to FACS approaches, as it circumvents limitations such as time requirements of FACS9. Instead, TRAP allows rapid and efficient isolation of RNA directly from bulk tissues, helping to avoid any alterations in gene expression. TRAP-seq is especially well-suited for use in the repopulating Fah-/- mouse liver, as hepatocyte expansion following removal of nitisinone is cell autonomous and enables gene expression profiling of the subset of hepatocytes with integrated transgenes. The TRAP vector can also be coexpressed with gene-activating or -silencing molecules17, including cDNA, short-hairpin RNA, and guide RNA, to study the effects on global gene expression of activation or inhibition of a specific gene. Alternatively, the RosaLSL-GFP-L10A transgenic mouse provides the ability to profile gene expression in any cell with Cre recombinase activity. Since GFP:RPL10A can be specifically expressed in any cells that express Cre, the role of other cell types in the liver during liver injury and repopulation could be studied. For instance, crossing CK19-Cre mouse with the TRAP transgenic mouse could be used to express GFP:RPL10A in cholangiocytes followed by TRAP-seq to study the change of gene expression in the biliary epithelium during the repopulation process.
To ensure accurate profiling of gene expression, it is critical to prepare all buffers and the affinity matrix prior to tissue dissection. All steps should be performed on ice with cold buffers unless otherwise specified to ensure polysome stabilization10 and prevent RNA degradation. All buffers should be prepared with RNase-free reagents and the TRAP-seq protocol should be carried out in an RNase-free environment to prevent RNA degradation and low yield of immunoprecipitated RNA. The affinity matrix can be prepared up to 2 weeks prior to use with gentle resuspension on a tube rotator overnight. Special care should be taken to not vigorously shake the matrix to prevent disruption of the antibody-conjugated, protein L-coated magnetic beads. The methods to prepare the affinity matrix includes conjugation of magnetic beads to biotinylated protein L followed by incubation with anti-GFP antibodies. However, commercially available protein A/G magnetic beads can be substituted; if used, skip the initial conjugation step and proceed directly to antibody binding. Furthermore, alternative epitope tags are presumably feasible with the above protocol with appropriate modification.
There are various points in which the RNA isolation and purification step can be paused (see protocol above). However, once liver samples have been harvested, continuing to the immunoprecipitation is recommended, as the yield of isolated RNA could drop by ~50% with freezing at this step10. Tissues should be quickly rinsed with dissection buffer that contains cycloheximide to inhibit mRNA translation. Insufficient tissue lysis could also contribute to low RNA yield. It is critical to homogenize tissues on ice until no tissue chunks are visible with the motor homogenizer while ensuring minimal aeration10. Additionally, sufficient washing with high-salt buffer is crucial to ensure removal of nonspecific binding of ribosomal proteins to the affinity matrix. Including a wild type negative control helps to assess the specificity of the immunoprecipitation and the efficiency of the wash steps. Additionally, using a commercial RNA purification kit that includes RNase-free DNase treatment will increase RNA purity.
Moreover, it is recommended to verify the expression and abundance of the GFP:RPL10A fusion protein and assess the amount of tissue required to obtain ample RNA for downstream analysis. Tissue sections or lysates could be used for immuno-based detection methods to validate the expression of GFP:RPL10A. The amount of RNA isolated can vary by: (1) the number of cells expressing GFP:RPL10A, (2) the expression level of the transgene, and (3) the size and ploidy of the cells expressing the transgene. A pilot experiment using half and double the amount of the recommended amount of tissue could be useful in determining the optimal input lysate for TRAP-seq. In our hands, we could obtain ~150 ng of RNA with as little as 1-2% of hepatocytes expressing GFP:RPL10A from 200 mg of the repopulating Fah-/- liver, representing ~2 x 105 polyploid hepatocytes with transgene expression9.
The TRAP-seq methodology isolates ribosome-bound mRNA to profile a cell&#39;s translating mRNA pool. The resulting sequencing reads therefore correspond to the &#39;translatome&#39; rather than the transcriptome. Note that translating ribosome footprints will not be collected, as TRAP is performed on native rather than crosslinked complexes. If footprinting analyses are desired, the above protocol should be modified with relevant cross-linking followed by immunoprecipitation (CLIP) methodologies18. Another limitation of TRAP is the requirement of a sufficient number of cells expressing the GFP:RPL10A fusion protein. For experiments in which the cell type of interest is small, combining multiple biological samples may be required to isolate sufficient RNA to enable RNA-seq19. Furthermore, TRAP-seq requires the presence of GFP:RPL10A in the cell type of interest. This could pose a challenge if there is no specific delivery system to the cells or if a cell-type specific promoter to drive Cre expression is not available.
The recent development of single-cell RNA-seq (scRNA-seq) technology has allowed direct sequencing followed by in silico identification of various cell types, enabling sequencing without sorting for specific cell types of interests20,21,22. However, scRNA-seq still requires dissociation of cells from the organ. In the case of the Fah-/- repopulation model, liver perfusion and hepatocyte isolation is extremely difficult and inefficient due to the fragility of both the injured and replicating hepatocytes. In fact, we have not yet been able to isolate sufficient hepatocytes from Fah-/- mice undergoing repopulation after hydrodynamic injection of Fah plasmids. Additionally, in the time it takes to process tissues, gene expression levels could change. Protocols for liver perfusion take up to 30 min of warm ischemia time. Future methodologies to optimize liver perfusion to decrease processing time and increase isolation efficiency could allow scRNA-seq integration to the Fah-/- mouse model system and possibly to other injury and repopulation models. This would also enable the study of all liver cell types.
In conclusion, the integration of TRAP-seq with the Fah-/- mouse allows specific isolation and gene expression profiling of replicating hepatocytes to identify therapeutic targets that could promote liver repopulation. This method can be implemented to study other cell types in the liver and other organ systems for disease-specific identification of gene expression changes to identify potential drug targets or biomarkers. An analogous technique can be used to collect nuclei from repopulating hepatocytes using affinity purification, and then to perform an epigenetic analysis of these specific cells16.

As the main metabolic organ in vertebrates, the liver is responsible for glucose homeostasis, serum protein synthesis, bile acid secretion, and xenobiotic metabolism and detoxification. The liver possesses an extraordinary capacity to regenerate the injured parenchyma upon exposure to toxins to prevent immediate liver failure1. However, failure of regeneration can occur in the setting of acetaminophen or alcohol overconsumption, which can lead to acute liver failure2. Furthermore, chronic liver injury caused by viral hepatitis infection, fatty liver disease, and steatohepatitis can cause liver fibrosis, cirrhosis, and hepatocellular carcinoma3. The only available curative treatment for end-stage liver disease is transplantation but is limited by organ shortage, preventing efficient treatment for all patients4. A better understanding of the recovery process after toxic liver injury is therefore crucial for the development of treatments to stimulate regeneration sufficient to rescue function in the diseased organ.
The most broadly applied model system for the study of liver regeneration is partial hepatectomy in rodents, in which a large proportion of the liver is resected to stimulate rapid hepatocyte expansion5. However, partial hepatectomy does not recapitulate hepatocyte expansion following toxic liver injury due to the lack of immune cell infiltration and hepatocyte cell necrosis often observed in the setting of acute liver injury in humans6. A more suitable system to model this form of organ renewal is the Fah-/- mouse, which lacks functional fumarylacetoacetate hydrolase (FAH) required for proper tyrosine metabolism and develops severe liver damage leading to death7. These mice can be maintained in a healthy state indefinitely by treatment with the drug nitisinone in the drinking water. Alternatively, FAH expression can be restored by transgene delivery to a subset of hepatocytes, which will expand to repopulate the liver upon nitisinone removal8.
To profile the gene expression changes of repopulating hepatocytes, a tool to specifically isolate replicating hepatocytes in the Fah-/- mouse without contamination from the neighboring injured hepatocytes and other cell types is required. Unfortunately, fluorescence-assisted cell sorting (FACS) of hepatocytes is difficult since (1) the fragility of repopulating hepatocytes leads to poor recovery after liver perfusion, (2) replicating hepatocytes are highly variable in size, making isolation of a pure population by FACS difficult, and (3) the procedure time from liver perfusion to RNA isolation is greater than 2 h, hence gene expression profiles may undergo substantial artificial changes before samples are acquired9.
Alternatively, the expression of epitope-tagged ribosomes specifically in repopulating hepatocytes allows for the rapid isolation of actively translating mRNA bound by ribosomes using affinity purification immediately after organ harvest, with bulk liver tissue lysates. Here, we describe a protocol to perform translating-ribosome affinity purification (TRAP)10 followed by high-throughput RNA-sequencing (TRAP-seq), to specifically isolate and profile mRNA in repopulating hepatocytes in the Fah-/- mouse9. Coexpression of green fluorescent protein-tagged ribosomal protein (GFP:RPL10A) with FAH allows affinity purification of translating mRNA bound by polysomes containing GFP:RPL10A. This method avoids any cell dissociation steps, such as liver perfusion to isolate fragile repopulating hepatocytes. Instead, it utilizes lysis of whole organ tissue and antibodies to rapidly extract the RNA specifically from the target cells. Finally, isolation of abundant, high-quality mRNA via TRAP-seq enables downstream applications such as sequencing analysis to profile the dynamic change of gene expression during the repopulation process.

<table><tbody><tr><td>10 mL Tissue Grinder, Potter-Elv, Coated</td><td>DWK Life Sciences (Wheaton)</td><td>358007</td><td></td></tr><tr><td>Absolutely RNA Miniprep Kit</td><td>Agilent</td><td>400800</td><td></td></tr><tr><td>Anti-GFP antibodies</td><td>Memorial Sloan-Kettering Antibody &amp;amp; Bioresource Core</td><td>GFP Ab #19C8 and GFP Ab #19F7</td><td></td></tr><tr><td>Bovine Serum Albumin, IgG-Free, Protease-Free</td><td>Jackson ImmunoResearch</td><td>001-000-162</td><td></td></tr><tr><td>cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td><td>Roche</td><td>11836170001</td><td></td></tr><tr><td>Cycloheximide</td><td>Millipore Sigma</td><td>C7698</td><td></td></tr><tr><td>Deoxycholic acid, DOC</td><td>Millipore Sigma</td><td>D2510</td><td></td></tr><tr><td>D-Glucose, Dextrose</td><td>Fisher Scientific</td><td>D16</td><td></td></tr><tr><td>DL-Dithiothreitol</td><td>Millipore Sigma</td><td>D9779</td><td></td></tr><tr><td>Dynabeads MyOne Streptavidin T1</td><td>Thermo Fisher Scientific</td><td>65602</td><td></td></tr><tr><td>Fisherbrand Petri Dishes with Clear Lid</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>HBSS (10x), calcium, magnesium, no phenol red</td><td>Thermo Fisher Scientific</td><td>14065-056</td><td></td></tr><tr><td>HEPES, 1 M Solution, pH 7.3, Molecular Biology Grade, Ultrapure, Thermo Scientific</td><td>Thermo Fisher Scientific</td><td>AAJ16924AE</td><td></td></tr><tr><td>Magnesium chloride, MgCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Millipore Sigma</td><td>M8266</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A452</td><td></td></tr><tr><td>NanoDrop 2000/2000c Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>VV-83061-00</td><td></td></tr><tr><td>NEBNext Poly(A) mRNA Magnetic Isolation Module</td><td>New England BioLabs</td><td>E7490S</td><td></td></tr><tr><td>NEBNext Ultra RNA Library Prep Kit for Illumina</td><td>New England BioLabs</td><td>E7530S</td><td></td></tr><tr><td>Nonylphenyl polyethylene glycol, NP-40. IGEPAL CA-630</td><td>Millipore Sigma</td><td>I8896</td><td></td></tr><tr><td>Nuclease-Free Water, not DEPC-Treated</td><td>Ambion</td><td>AM9932</td><td></td></tr><tr><td>Overhead Stirrer</td><td>DWK Life Sciences (Wheaton)</td><td>903475</td><td></td></tr><tr><td>PBS Buffer (10x), pH 7.4</td><td>Ambion</td><td>AM9625</td><td></td></tr><tr><td>Pierce Recombinant Protein L, Biotinylated</td><td>Thermo Fisher Scientific</td><td>29997</td><td></td></tr><tr><td>Potassium chloride, KCl</td><td>Millipore Sigma</td><td>P4504</td><td></td></tr><tr><td>RNA 6000 Pico Kit &amp;amp; Reagents</td><td>Agilent</td><td>5067-1513</td><td></td></tr><tr><td>RNaseZap RNase Decontamination Solution</td><td>Invitrogen</td><td>AM9780</td><td></td></tr><tr><td>RNasin Ribonuclease Inhibitors</td><td>Promega</td><td>N2515</td><td></td></tr><tr><td>Sodium azide, NaN&lt;sub&gt;3&lt;/sub&gt;</td><td>Millipore Sigma</td><td>S2002</td><td></td></tr><tr><td>Sodium bicarbonate, NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Millipore Sigma</td><td>S6297</td><td></td></tr><tr><td>SUPERase&amp;middot;In RNase Inhibitor</td><td>Invitrogen</td><td>AM2694</td><td></td></tr></tbody></table>

cell type-specific gene expression profiling in the mouse liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

To profile gene expression in repopulating hepatocytes of the Fah-/- mouse, Gfp:Rpl10a fusion and Fah transgenes are co-delivered within a transposon-containing plasmid8 (TRAP vector) to livers by hydrodynamic injection (Figure 1A). The removal of nitisinone induces a toxic liver injury that creates a selection pressure for hepatocytes stably expressing FAH to repopulate the injured parenchyma9. Immunofluorescence staining confirms the co-expression of FAH and the GFP:RPL10A fusion protein in repopulating hepatocytes after two weeks of liver repopulation (Figure 1B).
In the following representative experiment, TRAP-seq was performed using quiescent and repopulating mouse hepatocytes. First, to obtain GFP-tagged ribosomes from quiescent hepatocytes, transgenic RosaLSL-GFP-L10A mice were injected with AAV8-TBG-Cre 7 days prior to sacrifice to induce GFP:RPL10A expression in all hepatocytes14. We also processed a liver sample collected from a wild type mouse as a negative control to ensure isolation of translating mRNA was specific, meaning RNA could only be extracted from mice expressing GFP:RPL10A. The concentration of isolated RNA correlated with the number of cells expressing the fusion protein, with the quiescent sample producing the highest yield since all hepatocytes express GFP:RPL10A after AAV8-TBG-Cre injection (Figure 2A). Conversely, barely any RNA was detectable in wild type controls that did not have the GFP:RPL10A transgene, indicating the TRAP procedure is highly specific and has a low background. When TRAP was used on liver tissue undergoing repopulation with GFP:RPL10A-transduced hepatocytes, abundant, high-quality RNA was obtained (Figure 2B). In contrast, no RNA trace was detected via bioanalyzer for the negative control sample.
Downstream gene expression analysis can be carried out via reverse transcription and quantitative PCR or RNA-seq on TRAP-isolated RNA. Gsta1 encodes glutathione S-transferase that plays an important role for the metabolism of glutathione, the main detoxifying peptide to protect the liver against oxidative stress damage15. Gsta1 expression is induced by over 10-fold in repopulating hepatocytes as compared to quiescent hepatocytes, while no threshold cycle (Ct) value was detected with TRAP-isolated RNA from the wild type mouse due to the lack of input RNA (Figure 3A). Note that RNA quality can greatly impact the gene expression analysis. In the case of RNA-seq experiments, assessment of RNA quality should be performed according the recommendations of the library preparation kit and the sequencing platform (Figure 3B). A bioanalyzer is often used to determine the RNA integrity number (RIN), with a high RIN correlating with a higher rate of mRNA alignments to the genome (Figure 3B, left), whereas a lower RIN leading to a higher rate of ribosomal reads, indicating mRNA degradation (Figure 3B, right). Figure 3C,D demonstrates that TRAP-seq can identify differential gene expression in quiescent and repopulating hepatocytes. For instance, Alb expression is inhibited and Afp expression is upregulated during liver repopulation, reflecting that the replicating hepatocytes assume a less differentiated state to inhibit liver metabolic functions during repopulation9,16.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60242/60242fig1.jpg" /> 
Figure 1: Implementation of TRAP with Fah-/- to profile gene expression change of repopulating hepatocytes. (A) Schematic of expressing the GFP:RPL10A fusion protein with FAH in the Sleeping Beauty transposon system followed by injection into the Fah-/- mouse. Green hexagons indicate repopulating hepatocytes with stable expression of FAH and GFP:RPL10A, whereas black hexagons represent injured, dying hepatocytes. (B) Representative immunofluorescence staining demonstrates coexpression of GFP-tagged ribosomal protein L10A (green) and FAH (red) in the repopulating hepatocytes. Scale bar = 50 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60242/60242fig1large.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/60242/60242fig2.jpg" /> 
Figure 2: TRAP allows cell type-specific isolation of high-quality RNA. (A) The yield of RNA is positively correlated with the number of hepatocytes expressing GFP:RPL10A. The low yield of RNA from a wild type mouse demonstrates the specificity of TRAP from sources without the expression of GFP:RPL10A. (B) Bioanalyzer traces of total RNA isolated from repopulating livers expressing GFP:RPL10A and from wild type livers demonstrate the specificity of TRAP. Total RNA isolated from wild type liver tissue devoid of the GFP:RPL10A transgene shows that minimal RNA has been collected, whereas transgene-expressing tissue provides ample high-quality RNA. Note that ribosomal RNA peaks are present following successful TRAP10. FU, fluorescence unit. RIN, RNA integrity number. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60242/60242fig2large.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/60242/60242fig3.jpg" /> 
Figure 3: TRAP-isolated RNA can be used for downstream gene expression analysis. (A) Representative reverse transcription and quantitative PCR results of Gsta1 in quiescent and repopulating hepatocytes. No Ct value was detected with RNA isolated from wild type animals. (B) Alignment analysis of isolated RNA after high-throughput sequencing, demonstrating the importance of determining RNA integrity after isolation. High-quality RNA results in a higher percentage of mRNA reads (green), while low-quality RNA leads to a much higher percentage of ribosome reads (red), as most mRNA is degraded. RIN, RNA integrity number. (C,D) Integrative Genomics Viewer (IGV) tracks of RNA-seq reads of mRNA affinity-purified from quiescent and repopulating hepatocytes at the (C) Alb and (D) Afp loci. Note that the 3&#39; read bias is typical of a poly(A) selection pipeline. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60242/60242fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Cell Type-specific Gene Expression Profiling in the Mouse Liver at JoVE.com

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

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Gene Expression

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7.6K Views.  University of Pennsylvania. Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Video: Cell Type-specific Gene Expression Profiling in the Mouse Liver

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Genetics

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.

We present an in vitro mouse fetal liver erythroblast culture system that dissects the early and late stages of terminal erythropoiesis. This system facilitates functional analysis of specific genes in different developmental stages.

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony ...

Immunology and Infection

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.

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Developmental Biology

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

Profiling Genomic Histone Marks in Mouse Myoblasts Using CUT&#38;Tag

Using Cleavage Under Targets and Tagmentation (CUT&#38;Tag) Assay in Mouse Myoblast Research

This protocol paper aims to provide the new researchers with the full details of using Cleavage Under Targets and Tagmentation (CUT&amp;Tag) to profile the genomic locations of chromatin binding factors, histone marks, and histone variants. CUT&amp;Tag protocols function very well with mouse myoblasts and freshly isolated muscle stem cells (MuSCs). They can easily be applied to many other cell types as long as the cells can be immobilized by Concanavalin-A beads. Compared to CUT&amp;Tag, chromatin immunoprecipitation (ChIP) assays are time-consuming experiments. ChIP assays require the pre-treatment of chromatin before the chromatic material can be used for immunoprecipitation. In cross-linking ChIP (X-ChIP), pre-treatment of chromatin involves cross-linking and sonication to fragment the chromatin. In the case of native ChIP (N-ChIP), the fragmented chromatins are normally achieved by Micrococcal nuclease (MNase) digestion. Both sonication and MNase digestion introduce some bias to the ChIP experiments. CUT&amp;Tag assays can be finished within fewer steps and require much fewer cells compared to ChIPs but provide more unbiased information on transcription factors or histone marks at various genomic locations. CUT&amp;Tag can function with as few as 5,000 cells. Due to its higher sensitivity and lower background signal than ChIPs, researchers can expect to obtain reliable peak data from merely several millions of reads after sequencing.

Author Spotlight: Navigating Challenges and Innovations in Muscle Stem Cell Studies

Researchers new to the epigenetic field will find CUT&amp;Tag a significantly easier alternative to ChIP assays. CUT&amp;Tag has tremendously benefited the epigenetic studies on rare and primary cell populations, generating high-quality data from very few cells. This protocol describes performing H3K4me1 CUT&amp;Tag assays on mouse myoblasts isolated from mouse hindlimb muscles.

This protocol paper aims to provide the new researchers with the full details of using Cleavage Under Targets and Tagmentation (CUT&amp;Tag) to profile ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

Medicine

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Isolate Cell-Type-Specific RNAs from Snap-Frozen Heterogeneous Tissue Samples without Cell Sorting

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids potential pitfalls caused by the heterogeneity of tissues and unleashes the powerful transcriptome analysis. The protocol described here demonstrates how to use the Translating Ribosome Affinity Purification (TRAP) method to isolate ribosome-bound RNAs from a small amount of EGFP-expressing cells in a complex tissue without cell sorting. This protocol is suitable for isolating cell-type-specific RNAs using the recently available NuTRAP mouse model and could also be used to isolate RNAs from any EGFP-expressing cells.

This protocol aims to isolate cell-type-specific translating ribosomal mRNAs using the NuTRAP mouse model.

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

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Chapters in this video

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Using Cleavage Under Targets and Tagmentation (CUT&Tag) Assay in Mouse Myoblast Research

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Isolate Cell-Type-Specific RNAs from Snap-Frozen Heterogeneous Tissue Samples without Cell Sorting