Determination of Lsd1 function in C2C12 myoblast cells by global transcriptome analysis

First, we aimed to identify genes whose transcript levels change at the onset of myogenic versus adipogenic differentiation of muscle precursors. We therefore compared RNA-seq results obtained from C2C12 cells differentiated for 1 day in myogenic and adipogenic medium. Out of these genes, we wanted to determine those whose expression was affected by altered Lsd1 levels. To this end, we performed RNA-seq in C2C12 cells upon LSD1 overexpression and Lsd1 knock-down in adipogenic medium and compared them to corresponding control cells. Stable inducible LSD1 overexpressing C2C12 cell line was generated by infecting the cells with the lentivirus containing pSlik-Neo tetracycline-controlled transactivator (tTA) plasmid with Flag-tagged LSD1 construct downstream of the tetracycline-dependent promoter or empty vector and selecting with G418. To induce LSD1 overexpression, cells were treated with doxycycline (2 mg/ml) 48 h prior to differentiation and throughout the differentiation process. Lsd1 knock-down in C2C12 cells was induced by transfecting the cells with 20 nM siRNA against Lsd1 or unrelated control (Invitrogen) using DharmaFECT 3 reagent. Cells were differentiated for 1 day in adipogenic or myogenic medium and RNA was extracted with trizol. RNA integrity was confirmed by Bioanalyzer and cDNA library preparation and sequencing was performed (HiSeq 2000, paired-end, 100 bp). RNA samples were sequenced by the standard Illumina protocol to create raw sequence files (.fastq files). Reads were mapped to the mouse mm10 genome (NCBI Build 37) using TopHat version 2. The aligned reads were counted with the Homer software and differentially regulated genes were identified using EdgeR.