Permeability test
FITC labeled dextran with molecular weights of 40 kDa and 4 kDa was prepared at a concentration of 1 mg/ml using complete medium. The cells were washed twice with PBS and then treated with the respective FITC-dextran solutions. After one hour and two hours of treatment, 100 μL of medium from the lower layer was collected to measure the fluorescence intensity using a plate reader (Infinite® 200 PRO, Tecan, Switzerland).
RNA sequencing
Total RNAs were extracted using TRIzol Reagent and DNA digestion was performed using DNase I. The quality of RNA samples was assessed by measuring the A260/A280 ratio using the NanodropTM One spectrophotometer (Thermo Fisher Scientific Inc). Furthermore, RNA integrity was confirmed by conducting 1.5% agarose gel electrophoresis. Finally, qualified RNA samples were quantified using the Qubit3.0 with the QubitTM RNA Broad Range Assay kit (Life Technologies, Q10210). For stranded RNA sequencing library preparation, 2 μg of total RNA was utilized. The KC-DigitalTMStranded mRNA Library Prep Kit for Illumina® (Catalog NO. DR08502, Wuhan Seqhealth Co., Ltd., China) was employed, following the manufacturer’s instructions. This kit incorporates unique molecular identifiers consisting of 8 random bases to label the pre-amplified cDNA molecules. This approach helps to mitigate duplication bias during PCR and sequencing steps. Library products within the range of 200-500 bps were enriched, quantified, and subsequently sequenced on the DNBSEQ-T7 sequencer (MGI Tech Co., Ltd., China) using the PE150 model.