Apali

Genomic DNA was prepared from dissociated cardiomyocytes of the indicated mice. Genomic DNA from Ki67-CrexER cardiomyocytes was digested into 4.6 kb fragments by restriction enzyme ApaLI (NEB), while DNA from Ki67-Crex cardiomyocytes was digested into 3.6 kb fragments. The probe was designed based on the Cre region of the CrexER DNA sequence and the sequence was provided in the supplementary table 1. Shanghai Model Organisms Center, Inc. (SMOC) provided support in the Southern blotting experiments.

Taq polymerase (Sigma, St. Louis, MO) was used for PCR reactions to confirm presence of the plasmid using total DNA purified from C. thermocellum transformants as template. Reactions were performed with primers DC091 and DC508 according to the manufacturer's instructions (94 °C duplex denaturation, 56 °C annealing temperature, 1 min per kb at 72 °C for elongation). PCR products were visualized on a 1% agarose gel with an NEB 1 kb Ladder for size verification (NEB, Ipswich, MA). To verify structural stability in C. thermocellum, total DNA was electrotransformed into E. coli BL21 with a BioRad Gene Pulser (Biorad, Hercules, CA) using an exponential pulse in a 2mm cuvette (2.5 kV, 25 μF, 200 Ω). Selection in E. coli was performed with 50 µg/mL apramycin, and colonies were screened for the presence of the plasmid by performing restriction digests with EcoRI and ApaLI (NEB, Ipswich, MA).

A single transformant colony was picked directly into 20 mL LOD media lacking uracil, which was immediately degassed and incubated at 65°C. This strain maintaining the pJGW07 shuttle vector was named JWCH009. JWCH009 was grown to the late exponential phase (OD₆₈₀ approximately 0.15) in 50 mL LOD. Direct extraction of plasmid DNA from JWCH009 was performed as previously described [21 (link),25 (link)]. The plasmid DNA was digested with enzymes HaeIII, EcoRI, HhaI, and MboI (NEB).
To determine the structural stability of the plasmid, DNA was extracted from C. hydrothermalis with a gDNA Extraction kit (Zymo Research), and 2 μL of DNA was electrotransformed into E. coli DH5α via single electric pulse (2.5 kV, 200 Ω, and 25 microF) in a pre-chilled 2-mm cuvette using a Bio-Rad Gene Pulser. The cells were placed into 1 mL SOC media for 1 h with shaking at 37°C, and then plated onto LB agar supplemented with 50 μg/mL apramycin. The colonies were picked into 10 mL LB with 50 μg/mL apramycin, and the plasmid DNA was extracted using a Miniprep kit (Qiagen) and screened with restriction enzymes EcoRI and ApaLI (NEB) (Additional file 1: Figure S2B).

ϕX174 viral (+) strand (ssDNA) and ϕX174 replicative form I (dsDNA) were purchased from New England Biolabs. The ϕX174 replicative form I was digested by ApaLI (New England Biolabs) to generate linearized dsDNA. The supercoiled pBluescript DNA was purified from E. coli, according to manufacturer instructions using a commercially available Giga kit (Qiagen). All oligonucleotides were purchased from Integrated DNA Technologies. The oligonucleotides used in each assay were gel purified using denaturing polyacrylamide gel electrophoresis as described [43 (link)]. For the DNA binding assay, the H3 oligonucleotide (ssDNA) was 5'-radiolabeled with [³²P-γ]-ATP using T4 polynucleotide kinase [43 (link)]. To obtain the double-stranded substrate (dsDNA), ³²P-H3 was annealed to the unlabeled complementary H3c oligonucleotide. In the strand exchange assay, OL83-1 was used as the single-stranded substrate, and the double-stranded duplex DNA was generated by radiolabeling OL83-1, as described above, and annealing it with the unlabeled complementary oligonucleotide, OL83-2. The oligonucleotide OL90 was radiolabeled as described above and was used in the D-loop assay and nuclease protection assay. All previously mentioned oligonucleotides sequences can be found in Table 1.

Purified Rrp1 was incubated with cssDNA (circular single-strand DNA of Phi X174 viral DNA, NEB), ldsDNA (linear double-stranded DNA obtained by digesting Phi 174 RF I with ApaLI, NEB) or cccDNA (covalently closed-circular DNA of Phi 174 RF I, NEB) at 0.5 μM (base pair concentration) in E buffer (25 mM HEPES pH 7.5, 1 mM DTT, 60 mM KCl, 2 mM ATP, 3.5 mM MgCl₂, 5% glycerol) for 15 min in 37°C. Samples were then crosslinked with 0.2% glutaraldehyde (37°C, 5 min) then run on a 0.8% agarose 1xTAE gel and stained with SYBR Gold (ThermoFisher).

The assay is described in detail in supplementary text. Briefly, genomic DNA is digested with four restriction enzymes, AclI, ApaLI, EcoRI, and PciI, (New England Biolab, UK) that do not cut HTLV‐1, generating DNA fragments with an average size of 1000 bp. Digestion is followed by ligation of a linker (with the same overhang sequence) to the digested DNA. The sequences flanking the integration sites of the virus are then amplified using two HTLV‐1‐specific primers and two vectorette‐specific primers. The amplicons are detected by electrophoresis in a 1.5% agarose gel. The assay is conducted in triplicate.
A clone with a large number of cells will be detected in each of the triplicates and is reported as a dominant clone. A small clone will be detected inconsistently. A malignant clone may be detected as a single amplicon or as a dominant amplicon on a polyclonal background as shown in Figure S1.