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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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 remova...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#38; Bioresource Core</td>
			<td colspan="2">GFP Ab #19C8 and GFP Ab #19F7</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, 1M 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, MgCl2&#160;</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 &#38; 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, NaN3</td>
			<td>Millipore Sigma</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate, NaHCO3</td>
			<td>Millipore Sigma</td>
			<td>S6297</td>
			<td></td>
		</tr>
		<tr>
			<td>SUPERase&#183;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 injure...

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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7.5K 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

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

Spinal Cord Electrophysiology

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord dissection and preparation of recording bath artificial cerebrospinal fluid used for locomotor studies. Once dissected, the spinal cord ventral nerve roots can be attached to a recording electrode to record the electrophysiologic signals of the central pattern generating circuitry within the lumbar cord.

A demonstration of the isolation of neonatal mouse spinal cord for electrophysiologic studies.

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

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, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Heterokaryon Technique for Analysis of Cell Type-specific Localization

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling. These proteins display a diverse array of cellular functions including nuclear import/export of RNA and protein, transcriptional regulation, and apoptosis. Interestingly, major cellular reorganizations including cell division, differentiation and transformation, often involve such activities3,4,8,10. The detailed study of these proteins and their respective regulatory mechanisms can be challenging as the stimulation for these localization changes can be elusive, and the movements themselves can be quite dynamic and difficult to track. Studies involving cellular oncogenesis, for example, continue to benefit from understanding pathways and protein activities that differ between normal primary cells and transformed cells6,7,11,12. As many proteins show altered localization during transformation or as a result of transformation, methods to efficiently characterize these proteins and the pathways in which they participate stand to improve the understanding of oncogenesis and open new areas for drug targeting.
Here we present a method for the analysis of protein trafficking and shuttling activity between primary and transformed mammalian cells. This method combines the generation of heterokaryon fusions with fluorescence microscopy to provide a flexible protocol that can be used to detect steady-state or dynamic protein localizations. As shown in Figure 1, two separate cell types are transiently transfected with plasmid constructs bearing a fluoroprotein gene attached to the gene of interest. After expression, the cells are fused using polyethylene glycol, and protein localizations may then be imaged using a variety of methods. The protocol presented here is a fundamental approach to which specialized techniques may be added.

A flexible and efficient method for the characterization of cell type-specific protein localization and nucleocytoplasmic shuttling is described. This heterokaryon approach uses fluorescently-labeled fusion proteins to image protein localizations after cell fusion. The protocol is amenable to steady-state localizations or more dynamic determinations based on live cell imaging.

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...