This study was supported by Swedish Research Council (www.vr.se) grants to ARC (2014-6466 and the Swedish Foundation for Strategic Research (www.stratresearch.se) to ARC (ICA14-0060). Chalmers University of Technology provided financial support to MKME during this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This protocol is outlined in Figure 1 and includes the isolation of gDNA, digestion with restriction enzymes to be able to quantitate the number of ribonucleotides, treatment with alkali to hydrolyze the phosphodiester bonds of ribonucleotides incorporated into the gDNA, phosphorylation of free 5&#180;-OH ends, ssDNA ligation of adapters, second strand synthesis, and PCR amplification before sequencing.
1. Adapters and Index Primers
<ol>
	<li>Obtain ARC49, ARC140 oligonucleotides, ARC76/77, adapter and ARC78-ARC107 index primers (see Table 1). 
	NOTE: Oligonucleotides should be HPLC purified. ARC76/77 are ordered as duplex.</li>
	<li>Prepare 100 &#181;M stock solutions of each oligonucleotide in Tris-EDTA (TE) buffer (see the Table of Materials) and store at -20 &#176;C.</li>
	<li>Prepare 10 &#181;M solutions of ARC67/77 and 2 &#181;M solutions of ARC49 and index primers by diluting in elution buffer (EB; see the Table of Materials). Store at -20 &#176;C.</li>
</ol>
2. Growth and Harvest of Cells
<ol>
	<li>Grow HeLa cells in 70 mL Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum in a 250 mL Spinner flask at 37 &#176;C.</li>
	<li>Count the number of cells and collect 5x106 cells in a 50-mL tube, centrifuge for 5 min at 200 x g, and discard the supernatant.</li>
	<li>Wash the cells with 20 mL 1x PBS, centrifuge for 5 min at 200 x g, and discard the supernatant.</li>
	<li>Freeze the pellets at -20 &#176;C or continue with DNA purification.</li>
</ol>
3. DNA Purification and Quantitation
<ol>
	<li>Purify gDNA using phenol-chloroform extraction as described below.
	<ol>
		<li>Resuspend the cells in 2 mL lysis buffer (see the Table of Materials) and incubate for 30 min at 42 &#176;C on a heating block. 
		CAUTION: Lysis buffer contains hazardous components. SDS solution is irritating, Proteinase K is sensitizing, irritating, and toxic. Wear protective clothes and gloves.</li>
		<li>Split the sample in two 2 mL tubes and add 1 volume (V) of phenol-chloroform-isoamyl alcohol (25:24:1). 
		CAUTION: Phenol-chloroform-isoamyl alcohol is toxic, mutagenic, corrosive, and hazardous to aquatic environments. Use in a fume hood, wear protective clothes and gloves, and discard in special phenol-chloroform waste.</li>
		<li>Mix by inversion for 30-60 s and centrifuge for 5 min at 15,000 x g at room temperature. 
		NOTE: Do not vortex DNA to avoid introducing random strand breaks, which would distort the results.</li>
		<li>Transfer the upper, aqueous phase to a new 2 mL tube and add 1 V of phenol-chloroform-isoamyl alcohol (25:24:1).</li>
		<li>Mix by inversion and centrifuge for 5 min at 15,000 x g at 4 &#176;C.</li>
		<li>Transfer upper, aqueous phase to a new 2 mL tube and add 20 &#181;L NaCl (5 M) and 1 V of cold isopropanol. 
		CAUTION: Isopropanol is flammable, irritating, and toxic. Store it in a ventilated cabinet, wear protective clothes and gloves, and keep it away from flames.</li>
		<li>Mix by inversion and incubate for at least 1 h at -20 &#176;C.</li>
		<li>Centrifuge for 20 min at 15,000 x g, 4 &#176;C and discard supernatant.</li>
		<li>Wash DNA pellet with 200 &#181;L cold 70% ethanol, centrifuge for 20 min at 15,000 x g at 4 &#176;C and discard supernatant. 
		CAUTION: 70% ethanol is flammable and irritating. Keep working solution at -20 &#176;C, otherwise store in ventilated cabinet, wear protective clothes and gloves, and keep it away from flames.</li>
		<li>Dry the DNA pellet at room temperature for 20-25 min.</li>
		<li>Dissolve DNA pellets in 100 &#181;L TE buffer and pool the samples in one tube.</li>
	</ol>
	</li>
	<li>Quantitate DNA concentration using a dsDNA quantitation reagent according to manufacturer&#39;s specifications (see the Table of Materials). 
	NOTE: Use a dsDNA quantitation reagent, because spectrophotometric DNA quantitation can be affected by residual phenol.</li>
	<li>Store DNA at -20 &#176;C or continue with HincII treatment.</li>
</ol>
4. HincII Treatment and Alkaline Hydrolysis
<ol>
	<li>Digest 1 &#181;g of DNA in a reaction mix containing 5 &#181;L 10x buffer 3.1, 1 &#181;L (10 U) HincII, and nuclease-free H2O to a final volume of 50 &#181;L. 
	NOTE: To achieve optimal conditions for ligation, second strand synthesis and PCR amplification, it may be necessary to increase the amount of input DNA if it is expected that the DNA contains a very low number of ribonucleotides. Similarly, it may be necessary to decrease input DNA if the number of ribonucleotides is very high.</li>
	<li>Incubate for 30 min at 37 &#176;C.</li>
	<li>Purify HincII treated DNA with paramagnetic beads. 
	NOTE: Keep the tube lids open in the following steps to not disturb pellets by opening the tubes.
	<ol>
		<li>Add 1.8 V of paramagnetic beads to each sample, carefully mix by pipetting, and incubate at room temperature for 10 min.</li>
		<li>Use a magnetic rack to pellet the beads for 5 min, then remove and discard supernatant.</li>
		<li>Wash the pellet with 150 &#181;L of 70% ethanol (room temperature) for about 30 s then remove and discard the supernatant.</li>
		<li>Wash the pellet with 200 &#181;L of 70% ethanol (room temperature) for about 30 s then remove and discard the supernatant. 
		NOTE: Residual ethanol can be removed with a 10 &#181;L pipette. Droplets can be spun down briefly beforehand.</li>
		<li>Dry the samples at room temperature for around 15-20 min. 
		NOTE: The exact time depends on the volume of beads and the shape of the pellet, therefore the pellets should be checked visually.</li>
		<li>Remove tubes from the magnetic rack and elute pellet in 45 &#181;L EB, mix by pipetting carefully.</li>
		<li>Incubate for 5 min then pellet the beads on the magnetic rack and use 45 &#181;L of purified DNA in step 4.4.</li>
	</ol>
	</li>
	<li>Add 5 &#181;L of KOH (3 M) or KCl (3 M) to the DNA creating a total volume of 50 &#181;L. 
	CAUTION: 3 M KOH solution is corrosive. Wear protective clothes and gloves.</li>
	<li>Incubate for 2 h at 55 &#176;C in a hybridization oven followed by 5 min on ice. 
	NOTE: It is recommended to perform the KOH treatment in an oven rather than a heating block to maintain a uniform heating of the tube and prevent condensation at the lid.</li>
	<li>Precipitate DNA by adding 10 &#181;L sodium acetate (3 M, pH = 5.2) and 125 &#181;L cold 100% ethanol. Incubate on ice for 5 min. 
	CAUTION: 100% ethanol is flammable and irritating. Store in a ventilated cabinet, wear protective clothes and gloves, and keep away from flames.</li>
	<li>Pellet gDNA by centrifuging at 21,000 x g, 4 &#176;C for 5 min and discard the supernatant.</li>
	<li>Wash DNA pellet with 250 &#181;L cold 70% EtOH, centrifuge at 21,000 x g, 4 &#176;C for 5 min, and discard the supernatant. 
	NOTE: To remove droplets, the tube can be spun down briefly again and supernatant can be removed with a 10 &#181;l pipette.</li>
	<li>Let the pellet dry in an open tube for about 5-10 min until any visible fluid has evaporated.</li>
	<li>Let DNA pellet dissolve in 20 &#181;L EB for 30 min at room temperature.</li>
</ol>
5. 5&#180; End Phosphorylation
<ol>
	<li>Prepare the reaction mix for each sample in advance consisting of 2.5 &#181;L 10x T4 polynucleotide kinase reaction buffer, 1 &#181;L (10 U) 3&#180;-phosphatase-minus T4 polynucleotide kinase, and 2.5 &#181;L ATP (10 mM).</li>
	<li>Transfer 19 &#181;L of each DNA sample into a new 200 &#181;L tube and denature for 3 min at 85 &#176;C in a thermo-cycler.</li>
	<li>Cool DNA samples on ice and add 6 &#181;L of reaction mix to each sample.</li>
	<li>Incubate reaction mixes at 37 &#176;C for 30 min and stop the reaction by incubating the samples at 65 &#176;C for 20 min.</li>
	<li>Purify DNA as described in 4.3, using 1.8 V of paramagnetic beads but elute in 14 &#181;L EB.</li>
</ol>
6. ssDNA Ligation
<ol>
	<li>Prepare the reaction mix for each sample in advance consisting of 0.5 &#181;L ATP (2 mM), 5 &#181;L 10x T4 RNA ligase reaction buffer, 5 &#181;L CoCl3(NH3)6 (10 mM), 0.5 &#181;L ARC140 (100 &#181;M), and 25 &#181;L 50% PEG 8000. Mix well by pipetting. 
	CAUTION: CoCl3(NH3)6 is carcinogenic, sensitizing, and hazardous to aquatic environment. Wear protective clothes and gloves.</li>
	<li>Transfer 13 &#181;L of purified DNA from step 5.5 to a new 200 &#181;L tube and denature for 3 min at 85 &#176;C in a thermo-cycler.</li>
	<li>Cool the DNA on ice and add 36 &#181;L of reaction mix to each sample, mix by pipetting, and spin down briefly.</li>
	<li>Add 1 &#181;L (10 U) of T4 RNA Ligase to each reaction, mix by pipetting, and spin down briefly.</li>
	<li>Incubate the samples at room temperature in the dark overnight.</li>
</ol>
7. Second-strand Synthesis
<ol>
	<li>Purify ligated DNA as described in 4.3, but use 0.8 V of paramagnetic beads, pellet the beads for 10 min and elute in 20 &#181;L EB. 
	NOTE: Due to the higher viscosity of the ligation reaction mix, the first pelleting step is prolonged.</li>
	<li>Transfer 20 &#181;L of DNA sample to a new 200 &#181;L PCR tube. Repeat the purification step using 0.8 V of paramagnetic beads following the manufacturer&#39;s specifications and elute in 14 &#181;L EB.</li>
	<li>Prepare the reaction mix for each sample in advance consisting of 2 &#181;L of 10x T7 DNA polymerase reaction buffer, 2 &#181;L ARC76/77 (2 &#181;M), 2 &#181;L dNTPs (2 mM), and 0.8 &#181;L BSA (1 mg/mL).</li>
	<li>Transfer 12.8 &#181;L purified DNA to a new 200 &#181;L tube, denature for 3 min at 85 &#176;C in a thermo-cycler.</li>
	<li>Cool the DNA on ice and add 6.8 &#181;L of reaction mix to each sample, mix by pipetting, spin down briefly, and incubate for 5 min at room temperature.</li>
	<li>Add 0.4 &#181;L (4 U) T7 DNA polymerase to each reaction and incubate for 5 min at room temperature.</li>
	<li>Purify DNA as described in 4.3, using 0.8 V of paramagnetic beads and elute in 11 &#181;L EB.</li>
</ol>
8. PCR Amplification and Library Quantitation
<ol>
	<li>Prepare the reaction mix for each sample in a new 200 &#181;L tube in advance consisting of 7.5 &#181;L ARC49 (2 &#181;M), 7.5 &#181;L index primer (2 &#181;M, unique for each sample), and 25 &#181;L 2x hot start ready mix.</li>
	<li>Add 10 &#181;L of DNA sample to each reaction. Amplify the library using the following conditions: denature at 95 &#176;C for 45 s, followed by 18 cycles of 98 &#176;C for 15 s, 65 &#176;C for 30 s, 72 &#176;C for 30 s, ending with a final elongation at 72 &#176;C for 2 min. Hold samples at 4 &#176;C after amplification.</li>
	<li>Purify libraries as described in 4.3, using 0.8 V of paramagnetic beads, and elute in 20 &#181;L TE buffer.</li>
	<li>Quantitate libraries using a dsDNA quantitation reagent, according to the manufacturer&#39;s specifications (see the Table of Materials).</li>
	<li>Store samples at -20 &#176;C or continue with library analysis.</li>
</ol>
9. Library Analysis and Pooling
<ol>
	<li>Determine the quality of each library and estimate the average fragment size using a digital electrophoresis system. 
	NOTE: The average fragment size is assessed by estimating where the area under the curve of the electropherogram is halved, disregarding peaks from markers. Representative results of suitable library profiles after KOH or KCl treatment are given in Figure 2A.</li>
	<li>Calculate the concentration (nM) of the libraries as: 
	(c/103)/(p*650)]*109 
	where c is the concentration of the library in ng/&#181;L and p is the average fragment size in bp, as estimated in 9.1.</li>
	<li>Pool equal molar amounts of up to 24 libraries amplified with different index primers for sequencing. Add TE buffer to a final volume of 25 &#181;L and concentration of 10 nM. 
	NOTE: Depending on the number of libraries to be pooled, the amount of DNA from each library is adjusted. If primer dimers were detected in step 9.1 as a distinct peak of about 130 bp, the final volume of the library pool can exceed 25 &#181;L, because the purification step is repeated as described in 4.3, using 0.8 V of paramagnetic beads, and DNA is eluted in 25 &#181;L TE buffer.
	<ol>
		<li>Determine the new library pool concentration using a dsDNA quantitation reagent according to manufacturer&#39;s specifications and the average peak size as described above. Proceed to sequencing and data analysis (sections 10 and 11).</li>
	</ol>
	</li>
</ol>
10. Sequencing
<ol>
	<li>Perform 75-base paired-end sequencing on pooled libraries12.</li>
</ol>
11. Data Analysis
<ol>
	<li>Trim all reads to remove adapter sequences, filter for quality and read length. 
	NOTE: This can be done using cutadapt 1.2.120 with the command `cutadapt -f fastq --match-read-wildcards --quiet -m 15 -q 10 -a NNNNNNN &#60;FILE&#62;`, where NNNNNNN is replaced with the actual adapter sequence and &#60;FILE&#62; is replaced with the fastq file name.</li>
	<li>Remove mates of reads that were discarded in the previous step using custom scripts.</li>
	<li>Align Mate 1 of remaining pairs to an index containing the sequence of all oligonucleotides used in the library preparation (e.g., using Bowtie 0.12.821 and the command line options -m1 -v2). Discard all pairs with successful alignments.</li>
	<li>Align remaining pairs to the organism reference genome using Bowtie with the command line options -v2 -X10000--best.</li>
	<li>Map reads that span between the mitochondrial molecule beginning and end by aligning Mate 1 of all unaligned pairs (using Bowtie with the command line options -v2).</li>
	<li>Determine the count of 5&#180;-ends for all single and paired end alignments. Shift the position of these by one base upstream to the position where the hydrolyzed ribonucleotides were.</li>
	<li>Export data from the bowtie file format to a bedgraph file format using custom scripts for visualization in common genome browsers. Normalize the reads for each strand to reads per million.</li>
	<li>Using the position and counts from the bedgraph file, reference the organism genome sequence to determine the identity of incorporated ribonucleotides. 
	NOTE: For the human mitochondrial genome reads from the regions 16,200-300 and 5,747-5,847 for each strand should be excluded since these regions contain many free 5&#180;-ends unrelated to ribonucleotide incorporation by DNA polymerase &#947;.</li>
	<li>Divide the total reads, not including reads at the eleven HincII sites, with the mean number of reads per HincII site to get the number of ribonucleotides per single strand break, (i.e. the number of ribonucleotides per mitochondrial molecule).</li>
</ol>

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The authors declare that they have no competing financial interests.

Here we present a technique to simultaneously map and quantify ribonucleotides in gDNA, and mtDNA in particular, by the simple introduction of DNA cleavage at sequence specific sites in the genome as an addition to the established HydEn-seq protocol. While this study focuses on human mtDNA, originally the HydEn-seq method was developed in Saccharomyces cerevisiae, illustrating the method&#39;s translation to other organisms12,16.
For reliable results obtained from this approach, some critical steps should be noted: (A) Since sequencing adapters ligate to all available 5&#180;-ends, it is crucial to work with highly intact DNA. DNA should be isolated and libraries should be made preferably immediately after DNA isolation, or the DNA can be stored at -20 &#176;C. It is not recommended to store DNA in the fridge for a long time or to repeatedly freeze and thaw it. (B) To generate suitable libraries with this method, it is crucial to perform the KOH treatment of the DNA in an incubation oven, rather than a heating block, assuring homogenous heating of the whole sample and quantitative hydrolysis. (C) Furthermore, it is critical to control the quality of libraries before pooling and sequencing. The DNA should be quantified and analyzed using an automated electrophoresis system to ensure adequate amounts of library DNA, confirm appropriate fragment sizes, and check for primer dimers.
For a meaningful data analysis, it is also important to note that the informative value of this method is dependent on appropriate controls to assess background counts and sequence or strand biases. We routinely achieve a mapping efficiency in KCl samples of close to 70% when only digesting with the sequence specific endonuclease (Figure 2B, left panels). In addition, it is important to confirm that the endonuclease treatment is not affecting the overall detection of incorporated ribonucleotides by comparing HincII treated and untreated samples (Figure 3B). In these experiments, we have used HincII to introduce site specific cuts, though other high-fidelity restriction enzymes could also be used.
The protocol could be adapted to study other types of DNA lesions that can be processed to 5&#180;-phosphate or 5&#180;-OH ends. The accuracy of the results is dependent on the specificity of processing and requires suitable controls (e.g., wild type or untreated) for verification. Moreover, when adapting this method to other applications or for use with other organisms, one should consider that the method in its current setup requires about 1 &#181;g of DNA which is processed to a library. Since the number of ends is dependent on the number of embedded ribonucleotides, which varies depending on the organism or mutant, samples including a lower number of ribonucleotides would require more input DNA to generate a sufficient number of ends in the subsequent library construction. Similarly, if DNA samples have a much higher number of ribonucleotides, it would also require using less input DNA to obtain optimal conditions for ligation, second strand synthesis, and PCR amplification. It is noteworthy that the library construction as described in this protocol also generated data covering the nuclear genome (as displayed in Figure 2D) and only the data analysis was focused on mtDNA. This illustrates that larger genomes with moderately lower ribonucleotide frequencies are also captured by this method.
When considering this method, certain limitations should be taken into account: Although this method should, in theory, be applicable to virtually any organism, a suitable reference genome is necessary for the alignment of reads. Furthermore, the results obtained from our protocol represent the reads from a large number of cells. Specific ribonucleotide incorporation patterns of a subset of cells cannot be identified by this approach. If ribonucleotides are mapped in larger genomes with a very low number of ribonucleotides, it may be challenging to discriminate ribonucleotides from random nicks and appropriate controls are therefore needed.
The method we describe here, extends the available in vivo techniques such as HydEn-Seq16, Ribose-Seq17, Pu-Seq18, or emRiboSeq19. These approaches take advantage of the embedded ribonucleotides&#39; sensitivity to alkaline or RNase H2 treatment, respectively, employing Next-generation sequencing to identify ribonucleotides genome-wide, which allows their mapping and the comparison of relative incorporation. By cleaving the DNA sequence specifically, as described above, in addition to alkaline hydrolysis at embedded ribonucleotides, the reads for ribonucleotides can be normalized to those cleavage sites, allowing not only the identification and mapping of ribonucleotides, but also their quantitation for each DNA molecule. The application of our technique in the context of diseases related to DNA replication, DNA repair, and TLS could provide a deeper understanding of the role of ribonucleotides in underlying molecular mechanisms and genome integrity in general.

In a eukaryotic cell, the concentration of ribonucleotides (rNTPs) is much higher than the concentration of deoxyribonucleotides (dNTPs)1. DNA polymerases discriminate against ribonucleotides, but this discrimination is not perfect and, as a consequence, ribonucleotides instead of deoxyribonucleotides may be incorporated into genomes during DNA replication. Ribonucleotides may be the most common non-canonical nucleotides incorporated into the genome2. Most of these ribonucleotides are removed during Okazaki fragment maturation by RNase H2 initiated ribonucleotide excision repair (RER) or by Topoisomerase 1 (reviewed in reference3). Ribonucleotides that cannot be removed stay stably incorporated in the DNA2,4 and may affect it in both harmful and beneficial ways (reviewed in reviewed5). Besides being able to act as positive signals, for example in mating type switch in Schizosaccharomyces pombe6 and marking the nascent DNA strand during mismatch repair (MMR)7,8, ribonucleotides affect the structure9 and stability of the surrounding DNA due to the 2&#180;-hydroxyl group of their ribose10, resulting in replicative stress and genome instability11. The abundance of ribonucleotides in genomic DNA (gDNA) and their relevance in replication and repair mechanisms, as well as the implications for genome stability, give reason to investigate their precise occurrence and frequency in a genome-wide manner.
RNase H2 activity has not been found in human mitochondria and ribonucleotides are therefore not efficiently removed in mitochondrial DNA (mtDNA). Several pathways are involved in the supply of nucleotides to human mitochondria and to investigate whether disturbances in the mitochondrial nucleotide pool cause an elevated number of ribonucleotides in human mtDNA, we developed a protocol to map and quantitate these ribonucleotides in human mtDNA isolated from fibroblasts, HeLa cells, and patient cell lines12.
Most in vitro approaches (reviewed in reviewed13) to determine DNA polymerases&#39; selectivity against rNTPs are based on single ribonucleotide insertion or primer extension experiments where competing rNTPs are included in the reaction mix, allowing the identification or relative quantitation of ribonucleotide incorporation in short DNA templates. Quantitative approaches on short sequences may not reflect dNTP and rNTP pools at cellular concentrations and therefore provide insight into polymerase selectivity but are of limited significance regarding whole genomes. It has been shown that the relative amount of ribonucleotides incorporated during the replication of a longer DNA template, such as a plasmid, can be visualized on a sequencing gel using radiolabeled dNTPs and hydrolyzing the DNA in an alkaline milieu14. Furthermore, gDNA has been analyzed on Southern blots following alkaline hydrolysis, allowing strand-specific probing and determination of absolute rates of ribonucleotide incorporation in vivo15. These approaches allow a relative comparison of incorporation frequency but deliver no insight into the position or identity of the incorporated ribonucleotides. More recent approaches to analyze the ribonucleotide content in gDNA in vivo, like HydEn-Seq16, Ribose-Seq17, Pu-Seq18, or emRiboSeq19, take advantage of the embedded ribonucleotides&#39; sensitivity to alkaline or RNase H2 treatment, respectively, and employ Next-generation sequencing to identify ribonucleotides genome-wide. These methods do not provide insight into the absolute incorporation frequency of the detected ribonucleotides. By adding the step of sequence specific enzymatic cleavage to the HydEn-seq protocol, the method we describe here conveniently extends the information gained from a sequencing approach, allowing simultaneous mapping and quantitation of embedded ribonucleotides12. This method is applicable to virtually any organism given that highly intact DNA extracts can be generated and a suitable reference genome is available. The method could be adapted to quantitate and determine the location of any lesion that can be digested by a nuclease and leaves a 5&#180;-phosphate or a 5&#180;-OH end.
To map and quantitate ribonucleotides in genomic DNA, the method combines cleavage by a sequence specific endonuclease and alkaline hydrolysis generating 5&#180;-phosphate ends at sites where the specific recognition sequence for the endonuclease is located and 5&#180;-OH ends at positions where ribonucleotides were located. Since the generated free ends are subsequently ligated with adapters and sequenced using Next-generation sequencing, it is of importance to use highly intact DNA and avoid random fragmentation during DNA extraction and library preparation. Assessing these reads normalized to the reads at the endonuclease cleavage sites allows a simultaneous quantitation and mapping of the detected ribonucleotides. Free 5&#180;-ends are detected in control experiments where the alkaline hydrolysis of DNA is replaced by treatment with KCl. The acquired data provide insight into ribonucleotide location and quantity and allows analyses with respect to ribonucleotide content and incorporation frequency.

<table>
	<tbody>
		<tr>
			<td>10x T4 Polynucleotide Kinase Reaction Buffer</td>
			<td>New England Biolabs</td>
			<td>B0201S</td>
			<td></td>
		</tr>
		<tr>
			<td>10x T4 RNA Ligase Reaction Buffer</td>
			<td>New England Biolabs</td>
			<td>B0216L</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>Medicago</td>
			<td>09-9400-100</td>
			<td>dissolve 1 tablet in H2O to a final volume of 1 L</td>
		</tr>
		<tr>
			<td>2-Propanol</td>
			<td>Sigma-Aldrich</td>
			<td>33539-1L-GL-R</td>
			<td></td>
		</tr>
		<tr>
			<td>2100 Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2940CA</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube</td>
			<td>VWR</td>
			<td>525-0610</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-Triphosphate (ATP, 10 mM)</td>
			<td>New England Biolabs</td>
			<td>P0756S</td>
			<td>dilute with EB to 2 mM</td>
		</tr>
		<tr>
			<td>Agilent DNA 1000 Kit</td>
			<td>Agilent Technologies</td>
			<td>5067-1504</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA, Molecular Biology Grade (20 mg/mL)</td>
			<td>New England Biolabs</td>
			<td>B9000S</td>
			<td>diltue with nuclease-free H2O to 1 mg/mL</td>
		</tr>
		<tr>
			<td>Buffer EB</td>
			<td>QIAGEN</td>
			<td>19086</td>
			<td>referred to as EB</td>
		</tr>
		<tr>
			<td>CleanPCR paramagnetic beads</td>
			<td>CleanNA</td>
			<td>CPCR-0050</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxynucleotide (dNTP) Solution Mix (10 mM each)</td>
			<td>New England Biolabs</td>
			<td>N0447L</td>
			<td>dilute with EB to 2 mM</td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement</td>
			<td>Gibco</td>
			<td>61965026</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag 96 Side</td>
			<td>Thermo Fisher</td>
			<td>12331D</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 99.5% analytical grade</td>
			<td>Solveco</td>
			<td>1395</td>
			<td>dilute with milliQ water to 70%</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid solution (EDTA, 0.5 M)</td>
			<td>Sigma-Aldrich</td>
			<td>03690-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10500056</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES buffer pH 8.0 (1 M) sterile BC</td>
			<td>AppliChem</td>
			<td>A6906,0125</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexammine cobalt(III) chloride (CoCl3(NH3)6)</td>
			<td>Sigma-Aldrich</td>
			<td>H7891-5G</td>
			<td>dissolve in nuclease-free H2O for 10&#160;M solution, sterile filter. CAUTION: carcinogenic, sensitizing and hazardous to aquatic environment.</td>
		</tr>
		<tr>
			<td>HincII</td>
			<td>New England Biolabs</td>
			<td>R0103S</td>
			<td>supplied with NEBuffer 3.1</td>
		</tr>
		<tr>
			<td>Hybridiser HB-1D</td>
			<td>Techne</td>
			<td>FHB4DD</td>
			<td></td>
		</tr>
		<tr>
			<td>KAPA HiFi HotStart ReadyMix (2X)</td>
			<td>Kapa Biosystems</td>
			<td>KK2602</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysis buffer</td>
			<td></td>
			<td></td>
			<td>50 mM EDTA, 20 mM HEPES, NaCl 75 mM, Proteinase K (200 &#181;g/mL), 1% SDS</td>
		</tr>
		<tr>
			<td>Micro tube 1.5 mL</td>
			<td>Sarstedt</td>
			<td>72.690.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge 5424R</td>
			<td>Eppendorf</td>
			<td>5404000014</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge MiniStar silverline</td>
			<td>VWR</td>
			<td>521-2844</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiply&#160;&#181;StripPro 0.2 mL tube</td>
			<td>Sarstedt</td>
			<td>72.991.992</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>Ambion</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol &#8211; chloroform &#8211; isoamyl alcohol (25:24:1)</td>
			<td>Sigma-Aldrich</td>
			<td>77617-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>VWR</td>
			<td>26764.232</td>
			<td>dissolve in nuclease-free H2O for 3&#160;M solution, sterile filter</td>
		</tr>
		<tr>
			<td>Potassium hydroxide (KOH)</td>
			<td>VWR</td>
			<td>26668.296</td>
			<td>dissolve in nuclease-free H2O for 3&#160;M solution, sterile filter</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Ambion</td>
			<td>AM2546</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 3.0 Fluorometer</td>
			<td>Invitrogen</td>
			<td>Q33216</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Assay Tubes</td>
			<td>Invitrogen</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR Assay Kit</td>
			<td>Invitrogen</td>
			<td>Q32850</td>
			<td>CAUTION: Contains flammable and toxic components</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Invitrogen</td>
			<td>Q32851</td>
			<td>CAUTION: Contains flammable and toxic components</td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge 4K15</td>
			<td>Sigma Laboratory Centrifuges</td>
			<td>No. 10740</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS Solution, 10%</td>
			<td>Invitrogen</td>
			<td>15553-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate buffer solution, pH 5.2, 3 M (NaAc)</td>
			<td>Sigma-Aldrich</td>
			<td>S7899</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>VWR</td>
			<td>27810.295</td>
			<td>dissolve in nuclease-free H2O for 5&#160;M solution, sterile filter</td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-Rad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 Polynucleotide Kinase (3&#39; phosphatase minus)</td>
			<td>New England Biolabs</td>
			<td>M0236L</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 RNA Ligase 1 (ssRNA Ligase)</td>
			<td>New England Biolabs</td>
			<td>M0204L</td>
			<td>supplied with PEG 8000 (50%)</td>
		</tr>
		<tr>
			<td>T7 DNA Polymerase (unmodified)</td>
			<td>New England Biolabs</td>
			<td>M0274S</td>
			<td>supplied with 10x T7 DNA Polymerase Reaction Buffer</td>
		</tr>
		<tr>
			<td>TE Buffer</td>
			<td>Invitrogen</td>
			<td>12090015</td>
			<td></td>
		</tr>
		<tr>
			<td>ThermoMixer F2.0</td>
			<td>Eppendorf</td>
			<td>5387000013</td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous mapping and quantitation of ribonucleotides in human mitochondrial dna

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5&#180;-OH ends or 5&#180;-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5&#180;-end processing, DNA damage, and DNA repair.

Illustrating the methodology described above, representative data were generated analyzing human mitochondrial DNA from HeLa cells12. Figure 2B shows the summarized reads at all HincII sites in heavy (HS) and light strand (LS) of human mtDNA after KCl treatment (left panels). Around 70% of all detected 5&#180;-ends localize to the cut-sites, demonstrating the high efficiency of the HincII digestion. Treating libraries with KOH to hydrolyze the DNA at embedded ribonucleotides decreases the number of reads at HincII sites to about 40% (Figure 2B, right panels). This is expected since large numbers of 5&#180;-ends are generated at the sites of ribonucleotide incorporation, and is indicative of a sufficient library quality. Figure 2C illustrates the localization and frequency of 5&#180;-ends (green) after KCl treatment and reads generated by HydEn-seq (magenta) after KOH treatment, detecting both free 5&#180;-ends and ends generated at ribonucleotides by alkaline hydrolysis. Free 5&#180;-ends and ribonucleotides localizing to the HS of human mtDNA are shown in the left panel and those localizing to the LS are shown in the right panel. The relative numbers of raw reads at ribonucleotides (Figure 2D, upper panel) or HincII sites (lower panel) on HS and LS of mtDNA show, respectively, a 14-fold or 31-fold stronger coverage of the LS relative to the HS, while a similar bias was not observed for nuclear DNA. This strand bias may be explained by the distinct difference in base composition of the two strands and illustrates the importance of the normalization to reads at HincII sites.
Normalizing read counts to HincII gives a quantitative measure of the number of ribonucleotides per mitochondrial genome (Figure 3A). As illustrated in Figure 3B, the reads after KOH treatment for each ribonucleotide normalized to the sequence composition of each strand show a ratio different than 1, indicating a non-random distribution of reads suggesting a distinct ribonucleotide pattern and a high library quality. That ratio is unaffected by previous digestion with HincII, verifying the enzyme&#39;s cleavage specificity. Normalizing the reads at the sites of embedded ribonucleotides to those at HincII cleavage sites, as well as to the genome nucleotide content, generates a quantitative measure of how many of each ribonucleotide are incorporated per 1,000 complementary bases (Figure 3C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56551/56551fig1.jpg" /> 
Figure 1: Schematic for DNA Processing and Library Preparation. (1) Whole genomic DNA is cleaved by HincII for normalization in the subsequent quantitation of ribonucleotides, generating blunt ends at HincII sites (black arrowhead). (2) The DNA is treated with KOH to hydrolyze at ribonucleotide sites, leading to 2&#180;,3&#180;-cyclic phosphate (red pentagon) at 3&#180;-ends and free 5&#180;-OH ends. (3) 5&#180;-OH ends are phosphorylated by T4 Polynucleotide Kinase 3&#180;-phosphatase-minus. (4) All 5&#180;-ends carrying a phosphate group are ligated to the ARC140 oligonucleotide by T4 RNA ligase. (5) The second strand is synthesized using T7 DNA Polymerase and the ARC76-77 oligonucleotides containing random N6 sequences. (6) The library is amplified by a high-fidelity DNA Polymerase using ARC49 and one of the ARC78 to ARC107 index primers containing a unique barcode for multiplexing. (7) 5&#180;-ends are located by paired-end sequencing. <a href="https://cloudflare.jove.com/files/ftp_upload/56551/56551fig1large.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/56551/56551fig2.jpg" /> 
Figure 2: Method validation. (A) Representative electropherograms generated using an automated electrophoresis system to determine the quality of generated libraries treated with KOH or KCl. (B) Summarized signal at HincII sites in heavy (HS) and light strand (LS) human mtDNA after KCl (left panels) or KOH (right panels) treatment. (C) Circos figure of free 5&#180;-ends (green) and from HydEn-Seq (free 5&#180;-ends and ribonucleotides, magenta) in HS (left panel) and LS (right panel) human mtDNA. Peaks are normalized to per million reads and the maximum peak is adjusted to the maximum number of reads of the HydEn-seq library. (D) Summarized raw reads at ribonucleotides (upper panel) and HincII sites (lower panel) in heavy (H) and light (L) strand in human mtDNA (Mito.) or in reverse (RV) or forward (FW) strand in nuclear (Nuc.) DNA. Figures B, C and D are adapted from reference12. Error bars represent the standard error of the mean. <a href="https://cloudflare.jove.com/files/ftp_upload/56551/56551fig2large.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/56551/56551fig3.jpg" /> 
Figure 3: Representative Results. (A) The relative number of ribonucleotides normalized to reads at HincII sites for KOH treated libraries on the heavy (H) or light (L) strand. (B) Ratio of ribonucleotide identity to mtDNA genome composition for KOH treated (KOH) and HincII cleaved with KOH treated (HincII+KOH) libraries on the heavy (H) or light (L) strand of mtDNA. (C) Ribonucleotide frequency normalized to 1,000 complementary bases for HincII and KOH treated libraries on the heavy (H) or light (L) strand of mtDNA. Figures are adapted from reference12. Error bars represent the standard error of the mean. <a href="https://cloudflare.jove.com/files/ftp_upload/56551/56551fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td colspan="4">Sequence</td>
		</tr>
		<tr>
			<td>ARC49</td>
			<td colspan="4">AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC76</td>
			<td colspan="4">GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNN*N*N</td>
		</tr>
		<tr>
			<td>ARC77</td>
			<td colspan="4">AGATCGGAAGAGCACACGTCTGAACTCCAGTC*A*C</td>
		</tr>
		<tr>
			<td>ARC78</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC84</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC85</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC86</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC87</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC88</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC89</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC90</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC91</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC93</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC94</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC95</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC96</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATTGTTGACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC97</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATACGGAACTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC98</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATTCTGACATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC99</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATCGGGACGGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC100</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATGTGCGGACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC101</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATCGTTTCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC102</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATAAGGCCACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC103</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATTCCGAAACGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC104</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATTACGTACGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC105</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATATCCACTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC106</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATATATCAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC107</td>
			<td colspan="4">CAAGCAGAAGACGGCATACGAGATAAAGGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT</td>
		</tr>
		<tr>
			<td>ARC140</td>
			<td colspan="4">/5AmMC6/ACACTCTTTCCCTACACGACGCTCTTCCGATCT</td>
		</tr>
	</tbody>
</table>
Table 1: Oligonucleotides. Listed are the oligonucleotides used for HydEn-Seq. Bold face indicates indexing. * indicates a phosphorothioate bond. ARC140 contains a 5&#180;-amino group instead of a 5&#180;-OH group, in combination with a C6 linker. This modification reduces formation of ARC140 concatemers during ligation.

Watch this Scientific Journal Video about Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA at JoVE.com

Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5&#180;-end sequencing.

Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using ...

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9.4K Views.  University of Gothenburg. Here we describe a method amenable to simultaneously quantitate and genome-wide map ribonucleotides in highly intact DNA at single-nucleotide resolution, combining enzymatic cleavage of genomic DNA with its alkaline hydrolysis and subsequent 5´-end sequencing.

Video: Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.

Quantitation of Endothelial Cell Adhesiveness In Vitro

We report an in vitro method that allows the quantitation of the actual number of adhesive cells within an endothelial cell monolayer.

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This ...

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide&#8211;DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC Crosslinking of Small Molecules to Isolate Chromatin

Identifying the direct targets of genome-targeting molecules remains a major challenge. To understand how DNA-binding molecules engage the genome, we developed a method that relies on crosslinking of small molecules to isolate chromatin (COSMIC).

The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.