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Repeat elements analysis of Wild Type (WT) and Actb-/- (KO) Mouse Embryonic Fibroblasts using RNA-Seq

ABSTRACT: RNA sequencing of Wild Type (WT) and Actb-/- (KO) Mouse Embryonic Fibroblasts. Total RNA was sequenced to analyse noncoding transcripts and repeats

ORGANISM(S): Mus musculus

PROVIDER: GSE161964 | GEO | 2021/05/01

REPOSITORIES: GEO

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Next-generation sequencing analysis of chemically induced neurons from wild type (WT), Actb+/- (HET) and Actb-/- (KO) mouse embryonic fibroblasts (MEFs)

Project description:Purpose: The goals of this study are to use NGS to perform transcriptome profiling (RNA-seq) to find the difference of the transcriptomes of chemically induced neurons from wild type (WT), Actb+/- (HET) and Actb-/- (KO) mouse embryonic fibroblasts. Methods: MEFs were reprogramed into neurons by small chemical cocktails. Small chemical molecules were dissolved and diluted in DMSO and used at the following final concentrations: ISX9: 20 mM; Forskolin: 50 mM; CHIR99021: 20 mM; and I-BET151: 2 mM. In addition to the small molecules, the neuron induction medium (Neurobasal Medium) contains the following supplements: N2 (1X) and B27 (2X) supplements, GlutaMAX (1X), penicillin-streptomycin (100 µg/ml), bFGF (20 ng/ml), 100 µM cAMP, Non-essential Amino Acid (1X) and Trace element B (1X). MEFs were seeded to Matrigel-coated plate (1:30 dilution in pre-cold PBS and coat overnight at 4 oC, at a density of 200,000 cells per well in 6-well plate and 10,000 cells/well in 96 well plate. The MEFs were cultured in DMEM until confluent. When the cells are confluent, the DMEM was replaced with neuron induction medium with 4 small molecules. The induction medium was refreshed every two days for the first week and every 3 days for the remaining induction period until day 20. 3 biological replicates of total RNA were used for RNA-seq library preparation. Results: Using an optimized data analysis workflow, we identified neuronal gene programs induced during direct reprograming. The induction of neuronal programs were impaired in neurons from Actb-/- mouse embryonic fibroblasts. Conclusions: Our study shows that neuronal program induction was impaired in chemically induced neurons from Actb-/- mouse embryonic fibroblasts.

2018-04-27 | GSE113733 | GEO

Repeat elements analysis of Wild Type (WT) and Actb-/- (KO) Mouse Embryonic Fibroblasts using RNA-Seq

Project description:Repeat elements analysis of Wild Type (WT) and Actb-/- (KO) Mouse Embryonic Fibroblasts using RNA-Seq

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Actb knockout in primary mouse embryonic fibroblasts LC-MS/MS

Project description:The cytoplasmic actin proteins, beta- and gamma-actin, are 99% identical but perform non-redundant functions. Genes encoding the cytoplasmic actins, Actb and Actg1, respectively, are less similar but still share 89% of their nucleotide sequences. Knockout (KO) of Actb by deletion of first coding exons 2 and 3 in mice is embryonic lethal while KO embryonic fibroblasts (MEFs) fail to proliferate. In contrast, KO of Actg1 is viable but mice lacking Actg1 present with increased perinatal lethality and Actg1 KO MEFs present with a much milder defect in cell proliferation. Recent studies have identified important protein-independent functions for both Actb and Actg1 and demonstrate that deletions within the Actb nucleotide sequence, and not loss of the beta-actin protein, cause the most severe phenotypes in KO mice and cells. Here, we use a multi-omics approach to better understand what drives the phenotypes of Actb KO MEFs. RNA-sequencing and mass spectrometry of Actb KO MEFs reveal largescale changes to the transcriptome, proteome, and phosphoproteome. Pathway analysis of genes and proteins differentially expressed upon Actb KO shows widespread dysregulation of genes involved in the cell cycle. Together, these data suggest novel, protein-independent roles for Actb in regulating gene expression associated with control of cell proliferation.

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Xist-mediate chromatin inaccessibility requires B repeat

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ChIP-Seq of H3K27ac in Wild Type (WT), and Actb-/- (KO) Mouse Embryonic Fibroblasts

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