The slides were inserted into the slide box, and the tissue sections were separated into separate reaction chambers. For prepermeability, the sections were incubated at 37°C with 0.5 U/mL collagenase (ThermoFisher) and 0.2 mg/mL BSA (ThermoFisher) for 20 min in HBSS buffer (ThermoFisher). The wells were washed with 0.1 x SSC (Sigma‒Aldrich), dissolved with 0.1% pepsin (Sigma‒Aldrich) in 0.1 m HCL (Sigma‒Aldrich) and permeated at 37°C for 10 min. After incubation, the pepsin solution was removed and washed with 0.1 x SSC. After reverse transcription, Qubit3.0 was used for preliminary quantification, and the library was diluted to 1ng/ul. Then, Agilent 2100 was used to detect the insert size of the library. After the insert size met the expectation, Q-PCR was performed using StepOnePlus Real-Time PCR System to accurately quantify the effective concentration of the library (the effective concentration of the library was required to be > 10nM) and ensure the quality of the library. Qualified libraries were sequenced using the Illumina platform.