Project description:To investigate the effects of CEBPD on pancreatic ductal cell tumorigenicity, we established doxycycline-inducible MIA PaCa-2 cells for CEBPD over-expression, as well as non-control cells with an empty inducible construct. RNAseq was performed on these cells in the presence and absence of doxycycline and we showed that CEBPD activates gene signatures associated with cell motility.
Project description: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/EBPδ 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/EBPδ-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.
Project description:Cebpd is a transcription factor regulating cytokines. We determined the role of Cebpd in the regulation of transciptional output. We generated 4T1 breast cancer cells with CRISPR/Cas9 mediated Cebpd knockout. We treated control sgRosa26 and sgCebpd cells with vehicle or IFNgamma (20ng/ml) for 24 hours and isolated RNA for sequencing.