Puc19 plasmid

cDNA fragment for rps9 was cloned into pUC19 plasmid (Takara) using rps9-probeF: 5′-GGTACCTGTTCGAGCCTGACACGGAC-3′ and rps9-probeR:5′-TAATACGACTCACTATAGCAAGCACTGCTGAGTCCCT-3′. Probes for other genes were used as previously described (Danilova et al. 2011 (link); Ear et al. 2016 (link)). Whole mount in situ hybridization was performed as described (Thisse and Thisse 2008 (link)).
Embryos for TUNNEL staining were stored as described in whole mount in situ hybridization. Subsequent TUNNEL staining was performed on the embryos following the manufacturer’s protocol (Roche, In Situ Cell Death Detection Kit, TMR red).
For o-dianisidine staining, embryos were collected and fixed at room temperature for 2 h using 4% paraformaldehyde (PFA) in PBS buffer after dechorionation. After the removal of PFA by PBS wash, embryos is incubated in the o-dianisidine (Sigma) staining solution in the dark for 30 min followed by PBS wash. Embryos were then bleached for 20 min. Finally, embryos were immediately imaged after washed by PBS.

The ORF of the OsRZF1 gene without the stop codon was amplified (Supplemental Table S1) and cloned to the entry vector with a pENTR™/D-TOPO® Cloning Kit (Invitrogen, USA). Then the 2 × 35 s promoter (Marquès-Bueno et al., 2016 (link)) and amplified OsRZF1 ORF fused with EGFP marker were inserted into the pUC19 plasmid (Takara Bio Inc. Japan) using a Gibson assembly system (New England BioLabs, USA). Rice protoplasts were isolated and transformed according to the method previously reported (Saito et al. 2013 (link)). After 18 h, the EGFP fluorescence was detected using Zeiss LSM880 AxioObserver, equipped with an Argon laser as the excitation source, through a 20x/0.8 plan-Apochromat objective lens. EGFP was excited with an Argon laser at 561 nm and an intensity of 0.5% and detected between 597–624 nm with a detector gain value of 900. Images were analyzed and merged by ImageJ (NIH).

The plasma cfDNA from twenty patients of breast cancer was extracted to measure and evaluate the deamination efficiency of the optimized conversion method. To prove the finding a universal approach, we investigated whether the methylation status of the unmethylated CpG islands of MLH1 and MTND4P12 and the hyper-methylated CpG islands of RASSF1A in cancers [24 (link)] can be reproduced by our method in plasma from serious breast cancer patients. The primers of MTND4P12 and ASSF1A were shown in Table 1. The PCR product was purified and cloned into pUC19 plasmid (Takara Bio). Fifteen independent plasmid clones were picked up and subjected to sequence analysis.