RNA-seq analysis to examine the effect of Jmjd1a knockdown in 3T3-L1 pre-adipocyte cells

Adipocyte differentiation has been shown to require iron, but the underlying mechanism remains unclear. We have previously reported that iron supply through ferritinophagy is associated with demethylation of repressive histone marks in the differentiation-associated genes, which is mediated by the histone demethylase JMJD1A. To examine how JMJD1A regulates the gene expression program during adipocyte differentiation, in this study we peformed RNA-seq analysis using 3T3-L1 pre-adipocytes with Jmjd1a knockdown. 3T3-L1 cells were introduced with either control retroviral vector (shEmpty) or one expressing shRNA targeting Jmjd1a mRNA (shJmjd1a). For rescue experiments, either LacZ (as a control), wildtype (WT), or iron-binding deficient mutant JMJD1A (Mut) were expressed in shJmjd1a cells. These cells were then induced for adipocyte differentiation using the differentiation cocktail (insulin, DEX, and IBMX). On Day 2 of differentiaion, RNA-seq libraries were prepared as triplicate (n=3).