Simultaneous Mapping and Quantitation of Ribonucleotides in Human Mitochondrial DNA

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´-OH ends, ssDNA ligation of adapters, second strand synthesis, and PCR amplification before sequencing.

1. Adapters and Index Primers

1. 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.
2. Prepare 100 µM stock solutions of each oligonucleotide in Tris-EDTA (TE) buffer (see the Table of Materials) and store at -20 °C.
3. Prepare 10 µM solutions of ARC67/77 and 2 µM solutions of ARC49 and index primers by diluting in elution buffer (EB; see the Table of Materials). Store at -20 °C.

2. Growth and Harvest of Cells

1. Grow HeLa cells in 70 mL Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum in a 250 mL Spinner flask at 37 °C.
2. Count the number of cells and collect 5×10⁶ cells in a 50-mL tube, centrifuge for 5 min at 200 x g, and discard the supernatant.
3. Wash the cells with 20 mL 1x PBS, centrifuge for 5 min at 200 x g, and discard the supernatant.
4. Freeze the pellets at -20 °C or continue with DNA purification.

3. DNA Purification and Quantitation

1. Purify gDNA using phenol-chloroform extraction as described below.
   1. Resuspend the cells in 2 mL lysis buffer (see the Table of Materials) and incubate for 30 min at 42 °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.
   2. 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.
   3. 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.
   4. Transfer the upper, aqueous phase to a new 2 mL tube and add 1 V of phenol-chloroform-isoamyl alcohol (25:24:1).
   5. Mix by inversion and centrifuge for 5 min at 15,000 x g at 4 °C.
   6. Transfer upper, aqueous phase to a new 2 mL tube and add 20 µL 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.
   7. Mix by inversion and incubate for at least 1 h at -20 °C.
   8. Centrifuge for 20 min at 15,000 x g, 4 °C and discard supernatant.
   9. Wash DNA pellet with 200 µL cold 70% ethanol, centrifuge for 20 min at 15,000 x g at 4 °C and discard supernatant.
      CAUTION: 70% ethanol is flammable and irritating. Keep working solution at -20 °C, otherwise store in ventilated cabinet, wear protective clothes and gloves, and keep it away from flames.
   10. Dry the DNA pellet at room temperature for 20-25 min.
   11. Dissolve DNA pellets in 100 µL TE buffer and pool the samples in one tube.
2. Quantitate DNA concentration using a dsDNA quantitation reagent according to manufacturer's specifications (see the Table of Materials).
   NOTE: Use a dsDNA quantitation reagent, because spectrophotometric DNA quantitation can be affected by residual phenol.
3. Store DNA at -20 °C or continue with HincII treatment.

4. HincII Treatment and Alkaline Hydrolysis

1. Digest 1 µg of DNA in a reaction mix containing 5 µL 10x buffer 3.1, 1 µL (10 U) HincII, and nuclease-free H₂O to a final volume of 50 µL.
   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.
2. Incubate for 30 min at 37 °C.
3. 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.
   1. Add 1.8 V of paramagnetic beads to each sample, carefully mix by pipetting, and incubate at room temperature for 10 min.
   2. Use a magnetic rack to pellet the beads for 5 min, then remove and discard supernatant.
   3. Wash the pellet with 150 µL of 70% ethanol (room temperature) for about 30 s then remove and discard the supernatant.
   4. Wash the pellet with 200 µL of 70% ethanol (room temperature) for about 30 s then remove and discard the supernatant.
      NOTE: Residual ethanol can be removed with a 10 µL pipette. Droplets can be spun down briefly beforehand.
   5. 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.
   6. Remove tubes from the magnetic rack and elute pellet in 45 µL EB, mix by pipetting carefully.
   7. Incubate for 5 min then pellet the beads on the magnetic rack and use 45 µL of purified DNA in step 4.4.
4. Add 5 µL of KOH (3 M) or KCl (3 M) to the DNA creating a total volume of 50 µL.
   CAUTION: 3 M KOH solution is corrosive. Wear protective clothes and gloves.
5. Incubate for 2 h at 55 °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.
6. Precipitate DNA by adding 10 µL sodium acetate (3 M, pH = 5.2) and 125 µL 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.
7. Pellet gDNA by centrifuging at 21,000 x g, 4 °C for 5 min and discard the supernatant.
8. Wash DNA pellet with 250 µL cold 70% EtOH, centrifuge at 21,000 x g, 4 °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 µl pipette.
9. Let the pellet dry in an open tube for about 5-10 min until any visible fluid has evaporated.
10. Let DNA pellet dissolve in 20 µL EB for 30 min at room temperature.

5. 5´ End Phosphorylation

1. Prepare the reaction mix for each sample in advance consisting of 2.5 µL 10x T4 polynucleotide kinase reaction buffer, 1 µL (10 U) 3´-phosphatase-minus T4 polynucleotide kinase, and 2.5 µL ATP (10 mM).
2. Transfer 19 µL of each DNA sample into a new 200 µL tube and denature for 3 min at 85 °C in a thermo-cycler.
3. Cool DNA samples on ice and add 6 µL of reaction mix to each sample.
4. Incubate reaction mixes at 37 °C for 30 min and stop the reaction by incubating the samples at 65 °C for 20 min.
5. Purify DNA as described in 4.3, using 1.8 V of paramagnetic beads but elute in 14 µL EB.

6. ssDNA Ligation

1. Prepare the reaction mix for each sample in advance consisting of 0.5 µL ATP (2 mM), 5 µL 10x T4 RNA ligase reaction buffer, 5 µL CoCl₃(NH₃)₆ (10 mM), 0.5 µL ARC140 (100 µM), and 25 µL 50% PEG 8000. Mix well by pipetting.
   CAUTION: CoCl₃(NH₃)₆ is carcinogenic, sensitizing, and hazardous to aquatic environment. Wear protective clothes and gloves.
2. Transfer 13 µL of purified DNA from step 5.5 to a new 200 µL tube and denature for 3 min at 85 °C in a thermo-cycler.
3. Cool the DNA on ice and add 36 µL of reaction mix to each sample, mix by pipetting, and spin down briefly.
4. Add 1 µL (10 U) of T4 RNA Ligase to each reaction, mix by pipetting, and spin down briefly.
5. Incubate the samples at room temperature in the dark overnight.

7. Second-strand Synthesis

1. 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 µL EB.
   NOTE: Due to the higher viscosity of the ligation reaction mix, the first pelleting step is prolonged.
2. Transfer 20 µL of DNA sample to a new 200 µL PCR tube. Repeat the purification step using 0.8 V of paramagnetic beads following the manufacturer's specifications and elute in 14 µL EB.
3. Prepare the reaction mix for each sample in advance consisting of 2 µL of 10x T7 DNA polymerase reaction buffer, 2 µL ARC76/77 (2 µM), 2 µL dNTPs (2 mM), and 0.8 µL BSA (1 mg/mL).
4. Transfer 12.8 µL purified DNA to a new 200 µL tube, denature for 3 min at 85 °C in a thermo-cycler.
5. Cool the DNA on ice and add 6.8 µL of reaction mix to each sample, mix by pipetting, spin down briefly, and incubate for 5 min at room temperature.
6. Add 0.4 µL (4 U) T7 DNA polymerase to each reaction and incubate for 5 min at room temperature.
7. Purify DNA as described in 4.3, using 0.8 V of paramagnetic beads and elute in 11 µL EB.

8. PCR Amplification and Library Quantitation

1. Prepare the reaction mix for each sample in a new 200 µL tube in advance consisting of 7.5 µL ARC49 (2 µM), 7.5 µL index primer (2 µM, unique for each sample), and 25 µL 2x hot start ready mix.
2. Add 10 µL of DNA sample to each reaction. Amplify the library using the following conditions: denature at 95 °C for 45 s, followed by 18 cycles of 98 °C for 15 s, 65 °C for 30 s, 72 °C for 30 s, ending with a final elongation at 72 °C for 2 min. Hold samples at 4 °C after amplification.
3. Purify libraries as described in 4.3, using 0.8 V of paramagnetic beads, and elute in 20 µL TE buffer.
4. Quantitate libraries using a dsDNA quantitation reagent, according to the manufacturer's specifications (see the Table of Materials).
5. Store samples at -20 °C or continue with library analysis.

9. Library Analysis and Pooling

1. 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.
2. Calculate the concentration (nM) of the libraries as:
   (c/10³)/(p*650)]*10⁹
   where c is the concentration of the library in ng/µL and p is the average fragment size in bp, as estimated in 9.1.
3. 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 µL 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 µL, because the purification step is repeated as described in 4.3, using 0.8 V of paramagnetic beads, and DNA is eluted in 25 µL TE buffer.
   1. Determine the new library pool concentration using a dsDNA quantitation reagent according to manufacturer's specifications and the average peak size as described above. Proceed to sequencing and data analysis (sections 10 and 11).

10. Sequencing

1. Perform 75-base paired-end sequencing on pooled libraries¹².

11. Data Analysis

1. 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 <FILE>`, where NNNNNNN is replaced with the actual adapter sequence and <FILE> is replaced with the fastq file name.
2. Remove mates of reads that were discarded in the previous step using custom scripts.
3. 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.
4. Align remaining pairs to the organism reference genome using Bowtie with the command line options -v2 -X10000–best.
5. 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).
6. Determine the count of 5´-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.
7. 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.
8. 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´-ends unrelated to ribonucleotide incorporation by DNA polymerase γ.
9. 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).