Bsrgi

RNAi strain was constructed according to methods described by Choi and Schweizer (8 (link)) and Darsigny et al. (9 (link)). Five short hairpin RNA sequences targeting the sigX gene were designed and synthesized by Shanghai Generay Biotech Co., Ltd. (Shanghai, China) (Table S1 in Supplementary Material). After linearizing pCM130/tac vectors with the restriction enzymes NsiI and BsrGI (New England Biolabs, USA), the oligonucleotides were annealed and ligated to the linearized pCM130/tac vectors using T4 DNA ligase (New England Biolabs) following the manufacturer’s recommendations. The recombinant pCM130/tac vectors were transformed into the competent E. coli DH5a cells by heat shock and then were extracted and electroporated into P. plecoglossicida as described previously (10 (link)). Finally, the expression level of sigX of each RNAi strain was detected by qRT-PCR.

RNAi strain was constructed according to previously described methods (5 (link)). Five short hairpin RNA sequences targeting htpG, rplF, and flgD were designed and synthesized by Shanghai Generay Biotech Co., Ltd. (Shanghai, China) (Table S1). After linearizing pCM130/tac vectors with the restriction enzymes NsiI and BsrGI (New England Biolabs, U.S.A), the oligonucleotides were annealed and ligated to the linearized pCM130/tac vectors using T4 DNA ligase (New England Biolabs) based on the manufacturer's recommendations. The recombinant pCM130/tac vectors were transformed into competent E. coli DH5a cells by heat shock, and then extracted and electroporated into P. plecoglossicida. Finally, the expression level of the target gene of each RNAi strain was evaluated by qRT-PCR.

All plasmids were obtained from Addgene. The DAT was cloned into the Cry2–mCh plasmid to express Cry2–mCh at the N terminus of the DAT to ensure proper function because cloning of N-terminal–expressing YFP and FLAG to generate YFP–DAT and FLAG–DAT resulted in normal DAT function (12 (link), 35 (link), 38 (link)). The Cry2PHR–mCh (Addgene) vector was digested with BsrgI and NotI (New England Biolabs, Ipswich, MA). The DAT sequence was amplified from YFPsynDAT (Addgene), with flanking ends complementary to BsrgI (5′- TGA CCA TTG AAA CGG TAC CAC CAT GAG CAA GT-3′) and NotI (5′- TGC AAC TGC GGC CGC TCT CTT CAC ACC TTC AGC CAG T- 3′) sequences. DAT amplification products were isolated by gel electrophoresis and ligated into the digested Cry2–mCh vector using the Quick Ligation kit (New England Biolabs). The sequence was verified by sequencing and allele-specific polymerase chain reaction. Cry2–mCh–DAT (hereafter referred to as Cry2–DAT)–transformed DH5-alpha bacteria were cultured to propagate and isolate plasmids. We then isolated the plasmids by using Qiagen midiprep kit (Qiagen).

The percentage of single-stranded DNA (ssDNA%) generated by resection was measured as previously described (34 (link),35 (link)). Briefly, ER-AsiSI U2OS cells were treated with 5 μM 4-OHT for 4 h, and then genomic DNA (gDNA) was extracted with genomic DNA Extraction Kit (Tiangen Biotech,Beijing China). After that, 500 ng genomic DNA sample was digested or mock digested with 20 units of restriction enzymes BsrGI (New England Biolabs) at 37°C overnight. 1 μl DNA were used as templates in 10 μl of qPCR reaction containing 5 μl of 2× PerfectStart II Probe qPCR SupperMix (TransGen Biotech,Beijing, China), 0.2 μM of each primer and 0.2 μM probe. The sequences of qPCR primers and probes are shown in Supplementary Table S2. The ssDNA% generated by resection at selected sites was calculated with the following equation: ssDNA% = 1/(2^(△Ct –1) + 0.5) × 100.

End resection was assayed in ER-AsiSI cells that were predominantly in S/G2 phase by cell synchronization procedure mentioned earlier in the Methods section. The extent of resection adjacent to specific DSBs was measured by quantitative polymerase chain reaction (qPCR) as described previously [24 (link)]. The sequences of qPCR primers are shown in S1 Table. 20μL of genomic DNA sample (~200 ng in 1x CutSmart NEB restriction enzyme buffer) was digested or mock digested with 20 units of restriction enzymes (NmeAIII, AvaI, BsrGI, BamHI-HF or HindIII-HF; New England Biolabs) at 37°C overnight. 5 μl of digested or mock digested samples (~20 ng) were used as templates in 20 μl of qPCR reaction containing 10 μl of 2x iTaqUniversal SYBR Green Supermix, 500 nM of each primer using iCycler iQReal-Time PCR (Bio-Rad). The % ssDNA generated by resection at selected sites was determined as previously described [24 (link)]. Briefly, for each sample, a ΔCt was calculated by subtracting the Ct value of the mock-digested sample from the Ct value of the digested sample. The % ssDNA was calculated using algorithm: ssDNA% = 1/(2^{^}(ΔCt-1) + 0.5)*100 [24 (link)]. Data represent the mean of at least three independent experiments with SD values indicated by error bars.