Dna buffer set

Genomic DNA was isolated using the genomic-tip 20/G (Qiagen, #10223) and DNA Buffer Set (Qiagen, #19060). The detection of apurinic/apyrimidinic (AP) sites was performed using an aldehyde-reactive probe (ARP) kit (Kamiya Biomedical, #DN-002), according to the manufacturer's instructions. Data were expressed as the number of AP sites per 10⁶ nucleotides, as determined using a standard provided by the manufacturer.

Genomic DNA was isolated using the genomic-tip 20/G (Qiagen,#10223) and DNA Buffer Set (Qiagen, #19060). The detection of apurinic/apyrimidinic (AP) sites was performed using an aldehyde-reactive probe (ARP) kit (Kamiya Biomedical, #DN-002), according to the manufacturer's instructions.

The complete BP625 genome was sequenced using both short- and long-read sequencing methods. Genomic DNA was extracted using the Genomic-tip 100/G and DNA buffer set (Qiagen, Hilden, Germany). For short-read sequencing, a genomic library was prepared using the TruSeq DNA PCR-Free Kit (Illumina, San Diego, CA), followed by sequencing on the Illumina NovaSeq 6000 platform, generating 150-bp paired-end reads. Conversely, the long-read library was constructed using the SMRTbell Template Prep kit (Pacific Biosciences, Menlo Park, CA), and single-molecule real-time sequencing was performed using PacBio Sequel I (Pacific Biosciences, Menlo Park, CA). The Illumina platform yielded 6,411,695 reads, whereas the PacBio platform generated 176,974 subreads. All library preparation and sequencing steps were performed by Macrogen Co. (Tokyo, Japan). Before the downstream analysis, the raw read data were filtered using fastp version 0.20.1 for short reads (length of 20 bp; sequencing quality score of 20) [20 (link)] and Filtlong version 0.2.0 (https://github.com/rrwick/Filtlong) for long reads (length of 1,000 bp; 10% of poor-quality reads discarded).

The Sp (SZY643) and Sk (SZY661) strains are described by Nuckolls and Bravo Núñez et al. (Nuckolls et al. 2017 ). We obtained all other isolates from the National BioResource Center, Japan. We prepared genomic DNA using QIAGEN Genomic-tips (catalog number 10262 and 10243) using the QIAGEN DNA buffer set (catalog number 19060). We followed the kit protocol except that we extended the lyticase treatment to 18 h and the RNase A/Proteinase K treatment to 5 h. The Stowers Institute Molecular Biology core facility prepared the sequencing libraries using the Illumina Nextera Mate-Pair Sample Prep Kit (catalog number FC-132-1001). In total, 5- to 8-kb fragments were selected using a BluePippin machine (Sage Science). The libraries were sequenced (150-bp paired-end reads) on an Illumina MiSeq using the MiSeq Reagent Kit v2 (300 cycle) (catalog number MS-102-2002). Sequence data are available in SRA (accession number PRJNA476416).

We assessed nuclear and mtDNA damage as described previously [8 (link),12 (link),31 (link)]. Genomic DNA from cultured cells or paraffin-embedded lungs were extracted using the Qiagen Genomic Tip 20/G and Qiagen DNA Buffer set (Qiagen, Gaithersburg, MD, USA) according to the manufacturer’s protocol. Ex-Taq (Takara, Mountain View, CA, USA) was used for PCR, with specific primers to amplify a mitochondrial genomic in both short and long-form nuclear DNA (beta-globin) [8 (link),12 (link)]. For DNA quantification, we used PicoGreen (Thermo-Fisher/Invitrogen, Waltham, MA, USA) using the FL600 microplate fluorescence reader, with excitation and emission wavelengths of 485 and 530 nm, respectively. Mitochondria small fragment data were used to normalize fluorescence from the mitochondria long fragment. The number of mitochondrial lesions was calculated by the equation: