Next-generation sequencing of control (scramble) and Myo1c knockdown human podocytes, with and without TGF-β 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 TGFβ 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-β stimulation. Methods: Human control and Myo1c knockdown podocytes were differentiated for 14 days by thermoswitching from 33⁰C to 37⁰C 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-β in the same medium for a period of 48 hours. Control and TGF-β 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-β 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-β. Complex genetic network and genes effected due to Myo1c knockdown and TGF-β stimulation will provide a platform to define biological pathways in podocytes where Myo1c participates. Human podocytes mRNA profiles in control (scramble) and Myo1c knockdown cells were generated by deep sequencing, in triplicate, using Illumina. Contributor: Novogene Corporation