GSM4794742: RNA-Seq-shELK1-Induction-3T3-L1; Mus musculus; RNA-Seq
Total RNA was isolated using RNeasy Mini Kit (Qiagen) following the manufacturer's protocol. DNase I in column digestion was included to ensure the RNA quality. RNA-Seq analysis of global gene expression profiling was conducted at Anoroad (Beijing, China). Briefly, 3 µg RNA pooled from 3 different samples of the same treatment was used to generate sequencing libraries using UltraTM RNA Library Prep Kit for Illumina (NEB, #7530L). Sequencing was performed at 125 bp/150 bp paired-end reads on an Illumina Hiseq platform. Paired-end clean reads were aligned to the reference genome using Tophat (v2.1.1). To quantify the expression level of each gene, Cuffdiff (v2.2.1) was used to count the reads numbers mapped to each gene, and genes with a FPKM greater than 0.5 in any group were kept for further analysis. Genes which were induced more than 1.5 fold after the induction were considered as immediate-early responsive genes (IEGs) during adipogenesis. In DDB1 or ELK1 knockdown cells, IEGs with an induction fold less than 67% of those in the control cells were defined as DDB1 and ELK1 downstream genes, respectively. Gene ontology analysis were performed using the cluster Profiler R package.