Next-generation sequencing of control (scramble) and Myo1c knockdown human podocytes, with and without TGF-ß treatment

Purpose: Next-generation sequencing (NGS) was used to identify cellular pathways and genes through systems-based analysis. The goals of this study are to identify NGS-derived transcriptome profiling (RNA-seq) in control and Myo1c knockdown human podocytes upon TGFß stimulation. These high-throughput data were further validated through qRT–PCR methods to confirm the cellular pathways and genes affected due to genetic knockdown of Myo1c and TGF-ß stimulation. Methods: Human control and Myo1c knockdown podocytes were differentiated for 14 days by thermoswitching from 33°C to 37°C and removal of growth factors, insulin-transferrin-selenium from the medium. These podocytes were incubated overnight in RPMI medium with 0.1% FBS and stimulated with 5ng/ml of TGF-ß in the same medium for a period of 48 hours. Control and TGF-ß stimulated podocytes were processed for RNA isolation and submitted to Novogene for RNA-Seq. All the experiments were performed in triplicates. Conclusions: Our study is first to describe the detailed analysis of TGF-ß stimulated Myo1c knockdown podocyte transcriptomes using the RNA-seq technology. A comparative analysis of the differential expression profile was obtained between control vs. Myo1c knockdown podocytes, and control vs. Myo1c knockdown podocytes that were stimulated with TGF-ß. Complex genetic network and genes effected due to Myo1c knockdown and TGF-ß stimulation will provide a platform to define biological pathways in podocytes where Myo1c participates. Overall design: Human podocytes mRNA profiles in control (scramble) and Myo1c knockdown cells were generated by deep sequencing, in triplicate, using Illumina. Contributor: Novogene Corporation