Transcriptome analysis of FKBP25 siRNA-knockdown in cultured HEK 293 cells

Peptidyl-prolyl isomerases (PPIs) are a superfamily of ubiquitous enzymes that catalyze the cis-trans interconversion of Xaa-Pro peptide bonds. The functions of most PPIs remain uncharacterized. FKBP25, a mammalian PPI, localizes to the nucleus, binds nucleic acids, and associates with chromatin modifying enzymes. However, the role of this protein in transcriptional regulation remains unclear. To help address this question we present RNA-seq gene expression profiling in HEK293 cells transfected with siRNA targeting FKBP25 relative to a GFP-targeting negative control. Human embryonic kidney cells (HEK 293) were transfected with siRNA targeting FKBP25 or a GFP-targeting negative control and incubated for 72 h. Cells were harvested with trypsin, pelleted, and flash frozen before mRNA purification using the µMACs mRNA isolation kit (Miltenyl Biotec, Bergisch Gladbach, Germany). Purified mRNA was then analysed on an Agilent 2100 Bioanalyzer using Agilent 6000 RNA Nano Kit (Agilent Technologies, Santa Clara, California). cDNA was generated using the Superscript Double-Stranded cDNA Synthesis kit (ThermoFisher), 75bp paired-end libraries prepared using the Paired-End Sample Prep Kit (Illumina, San Diego, California) and then sequenced on a HiSeq 2000 sequencing system (Illumina).