Templiphi kit

Total DNA was extracted from dried leaf tissue using a modified CTAB method [43 (link)] and was used as a template for rolling-circle amplification (RCA) using φ29 DNA polymerase (TempliPhi kit, GE Healthcare, Buckinghamshire, UK). Amplified RCA products were digested with a set of restriction enzymes (HpaII, which recognizes a 4-nt site, and BamHI, EcoRI, HindIII, NcoI, NheI, and SalI, which recognize 6-nt sites). Restriction products were analyzed in 1% agarose electrophoresis gels in Tris-acetate-EDTA buffer that were stained with ethidium bromide and visualized under UV light. The selected digested RCA products (~2.7 and ~0.7 kbp) corresponding to putative full-length begomoviral genome components and deltasatellite genomes, respectively, were cloned into pBlueScript II SK (+) (Stratagene, La Jolla, CA, USA). Recombinant plasmid DNA was transformed into Escherichia coli DH5α by electroporation, and selected clones were sequenced by Macrogen Inc. (Seoul, South Korea).

Approximately 50 ng of DNA separated from heavy fractions in warming or control samples were amplified using a Templiphi kit (GE Healthcare, Little Chalfont, UK). The amplified DNA (2 μg) was labeled with fluorescent dye (Cy-3) dUTP using random primers and Klenow fragment of DNA polymerase I at 37 °C for 6 h, followed by heating at 95 °C for 3 min. Labeled DNA was then purified, dried in a SpeedVac at 45 °C for 45 min, and re-suspended in 43.1 μl of hybridization buffer containing 27.5 μl of 2X HI-RPM hybridization buffer, 5.5 μl of 10X CGH blocking agent, 2.4 μl of cot-1DNA, 2.2 μl of universal standard, and 5.5 μl of formamide. DNA was hybridized with GeoChip 5.0 (60 K) in a SL incubator (Shel Lab, Cornelius, OR, USA) at 67 °C for 24 h. Then, GeoChip arrays were washed and scanned by an MS 200 Microarray Scanner (Roche, Basel, Switzerland) at 532 nm and 635 nm. Raw signals from the scanning were processed by an online pipeline as previously described [58 (link)].

DNA was extracted from the spleen tissue from each of the ten Raja clavata. Briefly, approximately of 12 mg of tissue was homogenized with 300 µl of SM buffer (0.1 M NaCl, 50 mM Tris/HCl-pH 7.4, 10 mM MgSO₄) and disrupted using a bioruptor. The homogenized sample was centrifuged at 10.000 rpm for 2 min and 200 µl of the supernatant was used to isolate viral DNA using the High Pure Viral Nucleic Acid Kit (Roche Diagnostic, USA), according to manufacturer’s specifications. The extracted viral nucleic acid was then enriched for circular DNA molecules using the rolling circle amplification (RCA) reaction with the TempliPhi™ kit (GE Healthcare, USA). The products from RCA were quantified using Qubit™ dsDNA HS Assay kit (Thermo Fisher Scientific, USA), pooled equimolarly, and sent to Macrogen Inc. (Korea) for library preparation (Nextera DNA XT) and sequencing on an Illumina Novaseq 6000. Following Illumina sequencing, the resulting pair-end-reads were trimmed using Trimmomatic [31 (link)], host genome sequences were removed using the RefSeq genome of the Rajiform Amblyraja radiata available at NCBI (RefSeq accession number GCF_010909765.2) as a reference with Bowtie2 [32 (link)]. The remaining reads were de novo assembled using Megahit v1.2.9 [33 (link)].