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.