Cell Type-specific Gene Expression Profiling in the Mouse Liver

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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

1. Cycloheximide
   1. To make 500 µL of 0.1 g/mL cycloheximide, suspend 50 mg of cycloheximide in 500 µL 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 °C for up to 1 day. Cycloheximide inhibits translation.
2. Dithiothreitol (DTT)
   1. 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 °C. It is recommended to store 1 M DTT in single-use aliquots of 50 µL.
3. Deoxycholate (DOC)
   1. 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.
4. GFP antibodies
   1. Aliquot GFP antibodies when using for the first time. Snap freeze the aliquots and store at -80 °C.
      NOTE: It is recommended to store 50 µg of GFP antibodies in single-use aliquots.
5. B biotinylated protein L
   1. Resuspend biotinylated protein L in 1x phosphate-buffered saline (PBS) to make the final concentration 1 µg/µL.
      NOTE: Resuspended solution can be stored at -20 °C for up to 6 months.

2. Buffer Preparation

1. Bovine serum albumin (BSA) buffer
   1. 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 °C for up to six months.
2. Dissection buffer
   1. To make 50 mL of dissection buffer stock, combine 5 mL of 10x Hank's balanced salt solution (HBSS), 125 µL of 1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1,750 µL of 1 M glucose, and 200 µL 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 °C for up to six months.
   2. Immediately prior to use, add 100 µg/mL of 0.1 g/mL cycloheximide and keep on ice.
3. High-salt buffer
   1. To make 50 mL of high-salt buffer stock, add 1 mL of 1 M HEPES, 8.75 mL of 2 M KCl, 500 µL of 1 M MgCl₂, and 500 µL of 100% nonylphenyl polyethylene glycol (Table of Materials) to RNase-free water.
      NOTE: The high-salt buffer stock can be stored at 4 °C for up to six months.
   2. Immediately prior to use, add 0.5 µL/mL of 1 M DTT and 1 µL/mL of 0.1 g/mL cycloheximide. Keep the fresh high-salt buffer on ice.
4. Low-salt buffer
   1. To make 50 mL of low-salt buffer stock, add 1 mL of 1 M HEPES, 3.75 mL of 2 M KCl, 500 µL of 1 M MgCl₂, and 500 µL of 100% nonylphenyl polyethylene glycol to 44.25 Ml of RNase-free water.
      NOTE: The low-salt buffer stock can be stored at 4 °C for up to six months.
   2. Add 0.5 µL/mL of 1 M DTT and 1 µL/mL of 0.1 g/mL cycloheximide prior to use. Keep the fresh low-salt buffer on ice.
5. Tissue lysis buffer
   1. 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 µL of 1 M MgCl₂. Add RNase-free water to a final of 50 mL.
      NOTE: The dissection buffer stock can be stored at 4 °C for up to six months.
   2. Add 1 tablet/10 mL of EDTA-free protease inhibitor, 1 µL/mL of 0.1 g/mL cycloheximide, 10 µL/mL of RNase inhibitors each immediately prior to use. Keep the fresh tissue lysis buffer on ice.

3. Conjugation of Antibodies to Magnetic Beads

1. Antibodies
   1. Calculate the amount of GFP antibodies required for all samples and prepare for one extra sample.
      NOTE: For each sample, 50 µg of each GFP antibody is required.
   2. Thaw GFP antibodies on ice and spin at maximum speed (>13,000 x g) for 10 min at 4 °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.
2. Resuspend magnetic beads
   1. Resuspend magnetic beads (Table of Materials) by gentle pipetting.
   2. For each sample, use 150 µL 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.
   3. Collect beads on a magnetic stand for >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 >1 min and remove PBS.
3. Preparation of protein L-coated beads
   1. For each sample, use 60 µL of biotinylated protein L. If protein L is previously resuspended and stored at -20 °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.
   2. 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.
   3. Collect protein L-coated beads on a magnetic stand for >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.
   4. Collect coated beads on a magnetic stand for >1 min and remove the supernatant. Repeat the washing steps with 3% BSA for another 4 times (a total of 5 times).
4. Antibody binding
   1. Add the calculated amount of GFP antibodies into the protein L-coated beads and incubate for 1 h at 4 °C on a tube rotator.
      NOTE: After antibody incubation, take special care to not vortex the affinity matrix.
   2. During incubation, prepare low-salt buffer by calculating the total volume required for all samples and add 0.5 µL/mL of 1 M DTT and 1 µL/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 µL/sample for resuspension of the GFP-conjugated beads is required. Fresh low-salt buffer can be kept on ice for a couple of hours.
   3. Collect the affinity matrix on a magnetic stand for >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 >1 min and remove low-salt buffer. Repeat the washing steps with low-salt buffer for another 2 times (a total of 3 times).
   4. Resuspend the beads in low-salt buffer so that each sample has 200 µL of affinity matrix.
      NOTE: The affinity matrix can be stored in 0.02% NaN₃ at 4 °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 °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.

4. Liver Tissue Lysis

1. Buffer preparation and equipment setup
   1. 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.
   2. Prepare fresh dissection buffer by calculating the total volume required for all samples and add 1 µL/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.
   3. 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 µL/mL of 0.1 g/mL cycloheximide, and 10 µL/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.
   4. 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.
2. Repopulating liver homogenization
   1. Euthanize an 8−12-week-old Fah^-/- mouse injected with the TRAP vector and repopulated for 1−4 weeks with anesthesia and cervical dislocation according to approved animal experimental guidelines.
   2. 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.
   3. 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 °C after it is washed with dissection buffer. The protocol can be paused after this step.
   4. Weigh the liver on a Petri dish and Isolate 200−500 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.
   5. 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.
   6. Raise the speed to 900 rpm to fully homogenize the liver tissues for at least 12 full strokes.
   7. 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 °C. There could be a 50% decrease in isolated RNA if frozen lysates are used. The protocol can be paused after this step.
3. Nuclear lysis
   1. Centrifuge the liver lysate at 2,000 x g at 4 °C for 10 min and transfer the supernatant to a new, prechilled microcentrifuge tube on ice.
   2. 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.
   3. 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.
   4. Centrifuge the nuclear lysate at 20,000 x g at 4 °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.

5. Immunoprecipitation

1. 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 °C overnight, the same way as the immunoprecipitated samples are processed.
2. Add 200 µL 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 °C overnight with gentle mixing on a tube rotator.
   NOTE: The protocol can be paused for up to a day after this step.

6. RNA Isolation

1. Buffer preparation and equipment setup
   1. Place the magnetic rack at 4 °C for at least 30 min to pre-chill and keep the rack on ice throughout the experiment.
   2. Calculate the number of microcentrifuge tubes required and pre-chill on ice or at 4 °C.
      NOTE: Usually, each sample requires 1 microcentrifuge tube for the final purified RNA.
   3. 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 °C to compare with the bound fraction for transcript enrichment after purification. The protocol can be paused after this step.
   4. Prepare high-salt buffer by adding 0.5 µL/mL of 1 M DTT and 1 µL/mL of 0.1 g/mL cycloheximide to high-salt buffer stock.
      NOTE: For each sample, 5 mL of high-salt buffer is required.
2. RNA isolation
   1. 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.
   2. Collect beads on a magnetic stand for >1 min and remove the supernatant. Repeat the washing steps with high-salt buffer for another 4 times (a total of 5 times).
   3. Remove remaining high-salt buffer and remove microcentrifuge tubes from the magnetic stand and place at RT for 5 min to warm up.
   4. Resuspend the beads in 100 µL of lysis buffer (provided in the RNA isolation kit) with β-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.
   5. 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.
   6. Collect beads on a magnetic stand for >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 °C for up to 1 month before cleanup by warming up to RT upon thawing.
   7. 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 °C for maximum RNA recovery.
      NOTE: The isolated RNA can be stored at -20 °C for up to 1 month or -80 °C for several years. The protocol can be paused after this step.

7. Optional RNA Quality Analysis (Recommended)

1. 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.

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.

1. For TRAP-seq, perform RNA-seq library preparation according to standard methods¹³.
2. 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.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
10 mL Tissue Grinder, Potter-Elv, Coated	DWK Life Sciences (Wheaton)	358007
Absolutely RNA Miniprep Kit	Agilent	400800
Anti-GFP antibodies	Memorial Sloan-Kettering Antibody & Bioresource Core	GFP Ab #19C8 and GFP Ab #19F7
Bovine Serum Albumin, IgG-Free, Protease-Free	Jackson ImmunoResearch	001-000-162
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail	Roche	11836170001
Cycloheximide	Millipore Sigma	C7698
Deoxycholic acid, DOC	Millipore Sigma	D2510
D-Glucose, Dextrose	Fisher Scientific	D16
DL-Dithiothreitol	Millipore Sigma	D9779
Dynabeads MyOne Streptavidin T1	Thermo Fisher Scientific	65602
Fisherbrand Petri Dishes with Clear Lid	Fisher Scientific	FB0875712
HBSS (10x), calcium, magnesium, no phenol red	Thermo Fisher Scientific	14065-056
HEPES, 1 M Solution, pH 7.3, Molecular Biology Grade, Ultrapure, Thermo Scientific	Thermo Fisher Scientific	AAJ16924AE
Magnesium chloride, MgCl2	Millipore Sigma	M8266
Methanol	Fisher Scientific	A452
NanoDrop 2000/2000c Spectrophotometer	Thermo Fisher Scientific	VV-83061-00
NEBNext Poly(A) mRNA Magnetic Isolation Module	New England BioLabs	E7490S
NEBNext Ultra RNA Library Prep Kit for Illumina	New England BioLabs	E7530S
Nonylphenyl polyethylene glycol, NP-40. IGEPAL CA-630	Millipore Sigma	I8896
Nuclease-Free Water, not DEPC-Treated	Ambion	AM9932
Overhead Stirrer	DWK Life Sciences (Wheaton)	903475
PBS Buffer (10x), pH 7.4	Ambion	AM9625
Pierce Recombinant Protein L, Biotinylated	Thermo Fisher Scientific	29997
Potassium chloride, KCl	Millipore Sigma	P4504
RNA 6000 Pico Kit & Reagents	Agilent	5067-1513
RNaseZap RNase Decontamination Solution	Invitrogen	AM9780
RNasin Ribonuclease Inhibitors	Promega	N2515
Sodium azide, NaN3	Millipore Sigma	S2002
Sodium bicarbonate, NaHCO3	Millipore Sigma	S6297
SUPERase·In RNase Inhibitor	Invitrogen	AM2694