Se50 system

The total RNA was extracted from TGF-β1-treated HK-2 cells incubated with MSC+GW4869 (GW4869-1, GW4869-2, and GW4869-3) and treated with MSC-exo (exo-1, exo-2, and exo-3). RNA degradation and contamination, especially DNA contamination, were monitored on 1.5% agarose gels. RNA concentration and purity were measured using a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). RNA integrity was assessed using the RNA Nano 6000 Assay Kit and the Agilent Bioanalyzer 2100 System (Agilent Technologies, CA, USA). A total of 2.5 ng RNA per sample was used as the input material for RNA sample preparation. Sequencing libraries were prepared using NEBNext® Ultra™ small RNA Sample Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's instructions, and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kitv3-cBot-HS (Illumina) according to the manufacturer's instructions. After the cluster generation, sequencing was performed with an Illumina SE50 system at Biomarker Technologies Co., Ltd. (Beijing, China).

Total RNA was extracted from three samples as previously described [46 (link)] and used for sRNA and qRT-PCR assay. RNA quality and concentration were determined via 1.2% agarose gel electrophoresis and NanoDrop 2000c spectrophotometer (NanoDrop, Wilmington, DE, USA), respectively.
For construction and sequencing of the sRNA libraries, two replicates were performed for each sample. Firstly, sRNAs were isolated from total RNA by polyacrylamide gel electrophoresis (PAGE). Next, the isolated sRNAs were added to a 5′ RNA adaptor and a 3′ RNA adaptor by using T4 RNA ligase (TaKaRa, Dalian, China). Then, sRNAs with added 5′ and 3′ RNA adaptors were reverse transcribed into single-stranded cDNA using RT-PCR. Follows, the single-stranded cDNA was further synthesized into double-stranded cDNA by PCR amplification using adapter primers. Finally, the PCR product was purified and subjected to high-throughput sequencing by using the Illumina SE50 system at Biomarker Technologies Co., Ltd. (Beijing, China).