Quantstudio 7 flex machine

The fluorescence intensity tests were carried out on a 96-well reaction plate with the QuantStudio 7 Flex machine (Invitrogen Life Technologies, Carlsbad, CA, USA). FAM was excited at 495 nm, and its emission at 520 nm was recorded.
For Cas12a gate, the reaction was initiated by 50 nM LbCas12a and 50 nM gRNA with various concentrations of the target DNA and 1 μM FQ reporter substrates in 20-μl reaction volume [20 mM tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl₂, 1 mM DTT, 5% glycerol, and heparin (50 μg/ml)]. The reactions were performed in the 96-well PCR plates (100 μl) at 37°C for 4 hours with fluorescence measurements taken every 30 s.
For CONAN, 1 μM LbCas12a with 50 nM gRNA-T, 500 nM aDNA, and 1 μM scgRNA was mixed with various concentrations of the target DNA that were added directly into the 20-μl reaction volume. The reactions were performed in the 96-well PCR plate (100 μl) at 37°C for 4 hours with fluorescence measurements taken every 30 s.

qRT-PCR experiment was performed on a 96-well reaction plate with the QuantStudio 7 Flex machine (Invitrogen Life Technologies, Carlsbad, CA, USA) using a 2× SYBR premix Ex TaqTM GC kit. First, prepare 1:2 serially diluted DNA as standard. Then, the PCR reaction mixture was prepared in a volume of 10 μl, consisting of 5 μl of 1× SYBR premix, 2 μl of genomic DNA with the specific amount, 0.2 μl of 10 μM primer F, 0.2 μl of 10 μM primer R, and 2.6 μl of deionized water. The primers pair HBV-F/R used for PCR to detect HBV DNA were shown in table S4. Analyze melting curves of cccDNA PCR products to check specificity and standard curve to determine the PCR efficiency.
For NGS, the genomic DNA from six bladder tissue samples was sequenced with Illumina NextSeq 500 (Illumina, San Diego, CA, USA). In addition, the NGS dates were provided by Genetalks Biotech Co. Ltd. (Changsha, China). The immunohistochemical staining of the tissue sections from the normal bladder and bladder cancer was imaged under an Olympus CX31-LV320 (Olympus, Tokyo, Japan) bright-field microscope. In addition, these data were provided by the Third Hospital of Hebei Medical University (Shijiazhuang, China).