RNA-seq and genome-wide transcriptome analysis

Total sample RNA from renal tissues was isolated and purified using TRIzol (Thermofisher, 15,596,018), according to the manufacturer’s protocol. Total RNA was then quality controlled for quantity and purity with a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA) and checked for integrity with a Bioanalyzer 2100 system (Agilent, CA, USA); concentrations >50 ng/μL, RIN value > 7.0, and total RNA>1 μg are sufficient for downstream experiments. PolyA-bearing mRNA was specifically captured by two rounds of purification using oligo(dT) magnetic beads (Dynabeads Oligo(dT), cat. 25–61005, Thermo Fisher, United States). The captured mRNA was fragmented using the Magnesium Ion Fragmentation Kit (NEBNext^® Magnesium RNA Fragmentation Module, cat. E6150S, USA) at a high temperature of 94°C for 5–7 min. The fragmented RNA was synthesized into cDNA by reverse transcriptase (Invitrogen SuperScript™ II Reverse Transcriptase, cat. 1,896,649, CA, United States). Then, double-strand synthesis was performed using E. coli DNA polymerase I (NEB, cat. m0209, United States) and RNase H (NEB, cat. m0297, United States), and these complex duplexes of DNA and RNA were converted into DNA duplexes, while dUTP solution (Thermo Fisher, cat. R0133, CA, United States) was incorporated into the double-stranded DNA to blunt the ends of the double-stranded DNA. An A base is added at each end to allow it to be connected to a linker with a T base at the end, and the size of the fragments is screened and purified by magnetic beads. The second strand was digested with UDG enzyme (NEB, cat. m0280, MA, US) and then by PCR—pre-denaturation at 95 °C for 3 min, denaturation at 98 °C for a total of eight cycles of 15 s each, annealing to 60 °C for 15 s, extending at 72 °C for 30 s, and finally extension at 72 °C for 5 min to form a library with a fragment size of 300bp ± 50bp (strand-specific library). Finally, we pair-end sequenced it using the Illumina Novaseq™ 6000 system in the PE150 sequencing mode using standard procedures.