Agarose

To detect primary cytotoxicity of X-irradiation, DNA strand breaks were quantified by alkaline comet assay [17 (link)]. Cells were incubated with SAS (200 μM, 24h) prior to 1 Gy of X-irradiation. After irradiation, cells were suspended in 1% low-melting point agarose (Cat No. 317–01182; Nippon Gene, Tokyo, Japan) dissolved in serum free medium. Cell suspension containing 5×10³ cells were applied to microscope slide that pre-coated by 1% agarose (Cat No. 313–90231; Nippon Gene, Tokyo, Japan). Slides were incubated with comet lysis buffer (1.2 M NaCl, 100 mM EDTA, 0.1% sodium lauryl sarcosinate, 0.26 M NaOH) at 4°C for overnight. Then, slides were washed twice by alkaline buffer (0.03 M NaOH, 2 mM EDTA). Genomic DNA were separated in alkaline buffer at 9 V for 15min. After being washed with distilled water, DNA were stained by Propidium iodide (10 μg/mL, 4 °C, 30min). Fluorescent images were acquired using an BZ-X700 microscope (Keyence, Osaka, Japan), and comet tail were analyzed using Image J software with OpenComet plug-in at least 30 cells per sample [18 (link)].

A total of 52 primer pairs designed from flanking sequences surrounding intact MITEs (Yaakov et al., 2012 (link)) were used for the amplification of MITE fragments in this study (see Supplementary Table S1). PCR for amplification of MITE fragments was performed in a total reaction volume of 25 μl, containing 12.5 μl PCR Master Mix (Promega), 1.0 μl of genomic DNA (~50 ng/μl), 1.25 μl of each site-specific primer (6.1 pmol/μl) and 9.0 μl MilliQ water. The PCR was performed in a thermal cycler (GeneAmp PCR System 9,700, Applied Biosystems) using touchdown annealing temperature conditions as follows: initial denaturation at 94°C for 3 min; then 35 cycles with annealing decreasing by 2°C: 5 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 90 s; 5 cycles of 94°C for 1 min, 58°C for 1 min, 72°C for 90 s; 5 cycles of 94°C for1 min, 56°C for 1 min, 72°C for 90 s; followed by 20 cycles of 94°C for 1 min, 54°C for 1 min, 72°C for 90 s; and a final extension step at 72°C for 4 min. MITEs amplicons were size-separated using 1.5% agarose (Nippon gene, Japan) gel at 100 V for 25 min and stained with ethidium bromide. Stained gels were visualized under UV light and photographed using a gel documentation system.

In situ hybridization on paraffin wax-embedded sections of hagfish embryos from stages 45 to 53 (n = 1 biological replication) was performed as previously described [17 (link), 25 (link)]. Whole-mount in situ hybridization of lamprey embryos from stages 23 to 26 (n = 8 biological replicates) and shark embryos at stage 24 (n = 3 biological replicates) was performed according to Kusakabe et al. [48 (link)] and Sugahara et al. [49 ], respectively. As negative controls, sense strand probes were used for in situ hybridization in catshark and lamprey embryos. The hybridized lamprey embryos were fixed again and embedded in a mixture of 7.5% fish gelatin (Wako Pure Chemical Industries, Osaka, Japan) and 1.5% agarose (Nippon Gene); 40-μm-thick sections were obtained using a vibratome (LinearSlicer PRO7; Dosaka EM, Kyoto, Japan).