GSE69604
Identification of CEBPD regulated genes in MCF7 cells
Purpose: To determine which genes are regulated by CEBPD in MCF-7 cells. Methods: MCF7 cells were transfected with siRNAs against CEBPD or control. After 48h transfection total RNAs from two independent experiments were extracted and subjected to deep sequencing following Illumina standard protocols. Results: Samples were aligned to the reference human genome hg19 with good alignment ranging from 84 – 85% for both reads and very low mismatch rates <0.35%. RNA mapping statistics calculated using Picard software reported mapping of samples to 86 % mRNA bases, 63-65% unique reads, and <2.6% ribosomal reads. The aligned BAM files were imported into Partek Genome Studio v6.4 following the RNA-Seq workflow. Briefly, metadata about the samples was added followed by normalizing counts and doing differential expression analysis with ANOVA for the siNS vs siCEBPD contrasts. Differential gene list were created based on a p-value cutoff of <0.05 and fold-change of >1.5 or < -1.5. Analysis revealed that C/EBPd supports the expression of 319 genes (downregulated by siCEBPD) and attenuates the expression of 238 genes (induced by siCEBPD). For validation, 31 genes were assessed by QPCR with mRNA samples independent of those used for mRNA-Seq, and also by silencing CEBPD with either one of two siRNA sequences. About 90% of the tested genes from the mRNA-Seq approach were validated as C/EBPd-regulated in MCF-7 cells by QPCR. Conclusions: Our study is the first characterization of the CEBPD transcriptome generated by RNA-seq in MCF7 cells with biological replicates. Overall design: MCF7 cells were transfected with siRNA against CEBPD and control. 48h after total RNA was extracted followed by deep sequencing, in duplicate, using Illumina PE HiSeq2000.