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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)

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

PROVIDER: PRJNA453803 | ENA |

REPOSITORIES: ENA

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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

Next Generation Sequencing Analysis of chemically reprogrammed osteoblasts from Beta-actin Wildtype (WT) Heterozygotes (HET) and knockout (KO) Mouse Embryonic Fibroblast (MEFs)

Project description:mRNA profiles of chemically reprogrammed osteoblasts from Beta-actin Wildtype (WT) Heterozygotes (HET) and knockout (KO) Mouse Embryonic Fibroblast (MEFs)

2019-08-06 | GSE135392 | GEO

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.

2024-02-19 | PXD039786 | Pride

Genome-wide ChIP-Seq maps of EZH2 distribution between Wild Type (WT), Actb+/- (HET), and Actb-/- (KO) Mouse Embryonic Fibroblast

Project description:Genome-wide ChIP-Seq maps of EZH2 distribution between Wild Type (WT), Actb+/- (HET), and Actb-/- (KO) Mouse Embryonic Fibroblast

| PRJNA630705 | ENA

Next Generation Sequencing Analysis of chemically reprogrammed osteoblasts from Beta-actin Wildtype (WT) Heterozygotes (HET) and knockout (KO) Mouse Embryonic Fibroblast (MEFs)

Project description:Next Generation Sequencing Analysis of chemically reprogrammed osteoblasts from Beta-actin Wildtype (WT) Heterozygotes (HET) and knockout (KO) Mouse Embryonic Fibroblast (MEFs)

| PRJNA558732 | ENA

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

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

2021-05-01 | GSE161964 | GEO

Genome-wide ChIP-Seq maps of EZH2 distribution between Wild Type (WT), Actb+/- (HET), and Actb-/- (KO) Mouse Embryonic Fibroblast

Project description:β-actin is a crucial component of several chromatin remodeling complexes which control chromatin structure and accessibility. The mammalian Brahma-associated factor (BAF) is one such complex that plays essential roles in development and differentiation by regulating the chromatin state of critical genes and opposing the repressive activity of polycomb repressive complexes. While previous work has shown that β-actin loss can lead to extensive changes in gene expression and heterochromatin organization, it is not known if changes in β-actin levels can directly influence chromatin remodeling activities of BAF and polycomb proteins. Here we conduct a comprehensive genomic analysis of β-actin knockout mouse embryonic fibroblasts (MEFs) using ATAC-Seq, HiC-seq, RNA-Seq and ChIP-Seq of various epigenetic marks. We demonstrate that β-actin levels can affect the complex interplay between chromatin remodelers such as BAF/BRG1 and EZH2 in a dosage-dependent manner. Our results show that changes in β-actin levels and associated chromatin remodeling activities can not only impact local chromatin accessibility but also induce reversable changes in 3D genome architecture. Our findings support a novel role for β-actin levels in shaping the chromatin landscape during development and differentiation.

2021-05-01 | GSE149961 | GEO

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

Project description:Genome-wide ChIP-Seq of H3K27ac in WT and β-actin KO MEFs to assess the role of nulcear actin in regulating histone acetylation

2022-09-01 | GSE196089 | GEO

Single cell transcriptional analysis of chemically induced hepatocyte-like cells from mouse fibroblasts [scRNA-seq]

Project description:Here, we chemically induced hepatocyte-like cells (CiHeps) from mouse embryonic fibroblasts (MEFs) using a new protocol. To illustrate the transcriptomic landscape of hepatocyte cell formation by chemical induction, scRNA-seq was performed in C6F5UE-C6F(L)V-induced MEFs at 60 days of induction. The analysis of scRNA-seq data revealed that gastrointestinal-like and keratinocyte-like cells were induced along with CiHeps and these induced epithelial cells closely resembled that of their primary counterparts.

2022-08-09 | GSE210710 | GEO

RNA Sequencing Analysis of Nicol-Het, Nicol-KO and Ros1-KO Caput Epididymal Transcriptomes.

Project description:Purpose: The goal of this study is to detect differentially expressed genes, between Nicol +/- (Nicol-Het), Nicol-/- (Nicol-KO), and Ros1-/- (Ros1-KO) caput epididymis by RNA sequencing Methods: Caput epidiymal mRNA profiles of 14-week-old Nicol-Het, Nicol-KO, and Ros1-KO mice were generated by deep sequencing, in triplicate, using Illumina NovaSeq6000. Results: RNA-seq data identified differentially expressed transcripts. Conclusions: Our results show that the expression of many genes were downregulated in Nicol-KO caput epididymis compared with Nicol-Het one. The gene downregulation of Nicol-KO caput epididymis was similar to that of Ros1-KO one.

2023-02-27 | GSE206174 | GEO

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