Cw2298

The DNA of each sample was extracted according to the manufacturer's manual (CW2298; CWBio, China). The total RNA was extracted using TRIzol reagent according to the manufacturer's manual (Vazyme Biotech, China). HiScript reverse transcriptase (Vazyme Biotech, China) was used to process total RNA into cDNA, and PCR was performed in 50 μL of PCR buffer containing 3 μL of DNA (cDNA) template with each primer using the following steps: denaturation at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 40 s, annealing temperature as shown in Table S1 in the supplemental material for 40 s and elongation at 72°C for 90 s, ending with an additional elongation step of 10 min at 72°C. All of the primer pairs were referred to the previous reports (Table S1). PCR products were analyzed by 1% agarose gel electrophoresis, and the expected PCR products were purified and sequenced. qPCR was carried out in 20-μL reaction mixtures, which contained 10 μL of AceQ qPCR SYBR green master mix and 0.5 μL of each primer. The reactions were performed using the following conditions: 96°C for 3 min; 96°C for 30 s, 56°C for 60 s, 72°C for 90 s, 35 cycles; and 72°C for 10 min.

To identify the integration sites of the reporter cell line 3-B5-6, we performed whole genomic DNA sequencing. Genomic DNA of 3-B5-6 was extracted by using a universal genomic DNA kit (CWBio, CW2298). Whole genomic sequencing was performed by GENWIZ. Indels of human genome were mapped by pindel, which yields plasmid integration sites.