β-Actin-dependent global chromatin organization and gene expression programs control cellular identity.. Mus musculus

Purpose: The goals of this study are to use NGS to perform transcriptome profiling (RNA-seq) to find the difference of the gene expression programs among Wild Type (Wt), Actb+/- (Hetero) and Actb-/- (KNO) Mouse Embryonic Fibroblasts Methods: Total RNA was extracted from 70% confluent cells using TRI Reagent according to the manufacturer protocol (Sigma-Aldrich). Quality of total RNA was evaluated at SciLIfe lab (Stockholm, Sweden) using Qubit and Bioanalyzer respectively, samples which pass the QC were used for library construction according to the SciLife lab guideline using TruSeq Stranded mRNA Library Prep Kit (Illumina). Deep sequencing was performed at Science for Life Laboratory, the National Genomics Infrastructure, NGI, Karolinska Institute, Stockholm. Data was processed through the standard RNAseq analysis pipeline at NYUAD. Read aligmnet was performed using tophat2 v2.1.0 against Mus musculus GRCm38.p4 genome version. Cufflinks v2.2.1 was used to derive expression (FPKM) values. Cuffdiff was used to identify differential gene expression. Results: Using an optimized data analysis workflow, we identified genes differentially expressed between Wild Type and Actb+/-, WT and Actb-/-, Actb+/- and Actb-/- Mouse Embryonic Fibroblasts. Selected differentially expressed genes were confirmed by qRT-PCR. Gene ontology analysis further identified biological processes, cellular components and molecular functions that were overrepresented in the differentially expressed genes. Conclusions: Our study shows that there are gene programs differentially regulated among Wild Type, Actb+/- and Actb-/- Mouse Embryonic Fibroblasts Overall design: mRNA profiles of Wild Type (Wt), Actb+/- (Hetero) and Actb-/- (KNO) Mouse Embryonic Fibroblasts were generated by deep sequencing, in four biological replicates. One of Actb-/- sequencing sample that failed to pass initial QC analysis was excluded from differential gene expression analysis.