λ dna

Lyophilized proteins were dissolved into dissolving buffer and labelled with ATTO610-maleimide (ATTO-TEC) as described above, if necessary. λDNA (Takara) (26.25 µg) was attached to 1.65 µl DEAE sepharose beads (DEAE Sepharose Fast Flow, GE Healthcare) and stained with YOYO-1 (Thermo Fisher Scientific) if necessary in bead buffer (50 mM HEPES and 100 mM NaCl, pH 7.4). The bead suspension was then incubated with protein (0.8 μM) in a 96-well clear-bottom plate (Greiner Bio-One) at room temperature for 2.5 h. To examine the incorporation of LR domain-free RD, the ATTO610-labelled protein was added to a final concentration of 40 μM after 2.5 h of incubation of the DNA beads with LR-fused repeat protein. For the FRAP assay, ATTO610 signal on the beads was bleached using 561-nm laser light and observed by time-lapse imaging (FV3000, Olympus).

Tissue genomic DNA (200 ng) or urine total DNA (100 ng) were bisulfite converted using an EZ‐DNAm‐Gold Kit (Zymo Research Ltd., Cat. #D5006) and the DNA is dissolved in NF‐H₂O. For urine DNA less than 100 ng, λDNA (TaKaRa Ltd., Cat. #3010) was added to a total of 100 ng before bisulfite conversion to reduce DNA damage. After conversion, the DNA is amplified by the multiplex PCR system contained 20‐ng DNA, 2‐μl 10× Buffer II (100‐mM Tris–HCl, pH 8.3, 500‐mM KCl), 1.2‐μl 25‐mM MgCl₂, .4‐μl 10‐mM dNTPs, 4.6‐μl primer mix, .1‐μl AmpliTaq Gold DNA Polymerase (Thermo Fisher Ltd., Cat. #N8080241), 20‐ng DNA and NF‐H₂O to a total of 20 μl. Amplification was performed as follows: a denaturation step at 95°C for 10 min, followed by 25 cycles × (95°C for 30 s, 65°C for 30 s, 54°C for 2 min, 65°C for 30 s and 72°C for 30 s), 72°C for 10 min and 4°C for overnight. PCR products were purified with AMPure XP beads. Finally, the PCR products were amplified using adaptor oligos (Sangon Ltd., Shanghai, China) and KAPA HiFi HotStart ReadyMix (Roche Ltd., Cat. #KK2602), resulting in a final amplicon library that was further purified before sequencing by 150‐bp paired‐end format to a target of 6‐M reads on Illumina NovaSeq. A list of primers could be found in Table S8.

λ-DNA (TaKaRa, Co., Ltd. Dalian, China) was diluted in 1 M KCl at a pH of 8.0. All other chemical reagents used in the nanopore experiment were of analytical grade and use without further purification. The samples were prepared with Milli-Q super purified water with a resistance of >18 MΩ/cm. All solutions were filtered with a 0.02 μm Anotop filter (Whatman, Co. Maidstone, Kent, UK) before using.
The DNA was detected by nanopore sensors. A patch clamp amplifier (Axon Instruments, Axopatch 700B) was used to measure the corresponding ionic current flowing through the nanopore as a function of the biased voltages. The sampling frequency was above 100 kHz with a low pass filter of 10 kHz cutoff frequency. The current signals were recorded by the 1440A digitizer (Molecular Devices, Inc. CA, USA). Data were collected over multiple experiments with the same nanopore. The whole nanopore device was set in a Faraday cage for shielding electromagnetic noise.