Identification of CEBPD regulated genes in MCF7 cells
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ABSTRACT: 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:Reads were aligned to the mm10 genome with annotations provided by Ensembl. Transcripts were filtered, requiring at least 3 reads in 50% of samples within at least one condition. To identify differentially-regulated transcripts, we performed ANOVA (FDR-corrected p<0.05) and required the fold change to exceed 1.5.
Project description:The innate immune system is a two-edged sword; it is absolutely required for host defense against infection, but if left uncontrolled can trigger a plethora of inflammatory diseases. Here we used systems biology approaches to predict and validate a gene regulatory network involving a dynamic interplay between the transcription factors NF-κB, C/EBPδ, and ATF3 that controls inflammatory responses. We mathematically modeled transcriptional regulation of Il6 and Cebpd genes and experimentally validated the prediction that the combination of an initiator (NF-κB), an amplifier (C/EBPδ) and an attenuator (ATF3) forms a regulatory circuit that discriminates between transient and persistent Toll-like receptor 4-induced signals. Our results suggest a mechanism that enables the innate immune system to detect the duration of infection and to respond appropriately.
Project description:RNA was isolated from rectal biopsies from 190 pediatric patients undergoing diagnostic colonoscopy for inflammatory bowel diseases, including Crohn's disease and ulcerative colitis. Single-end, 75-bp sequencing was performed, and raw reads aligned to the human genome using Gencode v 24 as reference. We included 14085 protein-coding mRNA genes in downstream analyses, where cutoffs of fold change>1.5 and FDR<0.05 were considered significant.
Project description:To determine the potential molecular mechanisms by which STAT5 signaling control ileal Paneth cell homeostasis, we isolated total RNA from ileal intact crypts of STAT5+/+, STAT5DIEC-/- and STAT5DIEC+++ mice and performed RNA sequencing (RNA-seq). With an average of 22.3 million reads per sample, we observed 27540 transcripts when reads were aligned to the mm10 genome with annotations provided by Ensembl. Transcripts were filtered, requiring at least 3 reads in 50% of samples within at least one condition, leaving 10197 transcripts for analysis. To identify differentially-regulated transcripts, we performed ANOVA (FDR-corrected p<0.05) and required the fold change to exceed 1.5.
Project description:Purpose: The goal of this RNA sequencing (RNA-seq) study is to identify aberrations in the astrocyte transcriptional landscape caused by R270X mutation in MECP2. Methods: mRNA-seq analysis was performed on total RNA extracted from human embryonic stem cell (ESCs)-derived wild-type (WT) and MECP2-R270X mutant astrocytes. The R270X mutation was inserted into ESCs (line H1) via CRISPR/Cas9 technology. Samples were generated in triplicates, sequenced by Illumina NovaSeq 6000 Sequencing System. Raw reads were first trimmed for 10 bases at the 5’end to remove reads with biased nucleotide (ACGT) distribution. Trimmed reads were then aligned to the Homo sapiens genome (GRCh38p12, GENCODE, primary assembly), using STAR aligner. Differential gene expression (DEG) analyses on the read counts were performed using DESeq2. Genes with sum of read counts across all samples with less 10 were filtered out from analysis. Results: Our RNA-seq analysis showed that 1,621 genes were dysregulated in mutant astrocytes (fold-change >1.5 or <2/3; padj < 0.05, average reads count >10 in at least one genotype)
Project description:We examined if pediatric AMLs rank-ordered according to C/EBPδ expression showed the activation of similar pathways. AML samples were dichotomized into groups including the upper quartile (Q1) and the lower three quartiles (Q2-4) according to their C/EBPδ expression values. Moreover, AML samples were associated to French-American-British (FAB) classification.
Project description:We report the application of bioengineered skeletal muscle composed of murine skeletal myoblasts interspersed with human vascular endothelial cells (ECs) using spatially patterned scaffolds for the treatment of volumetric muscle loss. To gain insights into the molecular mechanisms by which spatial patterning promoted myo-angiogenesis, we performed mRNA sequencing of engineered muscle and endothelialized engineered muscle on either randomly-oriented or aligned scaffolds. The goals of this study are to compare NGS-derived transcriptome profiling (RNA-seq) from four groups: 1) randomly-oriented scaffold seeded with myoblasts; 2) randomly-oriented scaffolds seeded with myoblasts + ECs; 3) aligned scaffold seeded with myoblasts only; 4) aligned scaffold seeded with myoblasts + ECs. Samples were generated in triplicate and the NovaSeq 6000 (Illumina) sequencing platform was utilized. 150-bp paired-end reads were generated (20-30 million reads per sample). The raw data were checked for quality with FastQC (Version 0.11.7) and results were aggregated with MultiQC and were aligned to the mouse genome (GRCm38) using STAR (Version 2.5.3a) with ENCODE options for long RNA-seq pipeline. The alignment results were assessed using Samtools and aggregated with MultiQC (Version 1.5) and the differential gene expression analysis of the uniquely mapped reads/raw counts wwas performed using the DESeq2 package (Version 1.20.0). Each DE analysis was composed of a pairwise comparison between an experimental group and the control group. Differentially expressed genes were identified after false discovery rate (FDR = 0.05) correction.
Project description:We have performed sucrose-gradient-based isolation of polysomal fractions from untreated and TGF-beta treated MCF-10A and MCF7 cells, subjected these fractions to RNA-seq, and also sequenced total mRNA from each cell line in the treated and untreated condition