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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: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE113733 | GEO | 2018/04/27

REPOSITORIES: GEO

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Project description:Cortical rat neuronal culture, lysis and proteolytic digestion Primary rat cortical neurons were generated from E18 Sprague-Dawley rat embryos with minor modifications (Swanger, Mattheyses et al. 2015, Henderson, Greathouse et al. 2019). Neurons were cultured at a density of 4x105 cells per well in 12-well culture plates (Fisher Scientific, catalog no. 353043). Neurons were cultured in Neurobasal medium (Fisher Scientific, catalog no. 21103-049) supplemented with B27 (Fisher Scientific, catalog no.17504-044). Culture maintenance included a half media change every 2-3 days. At DIV 14, neurons were either treated with 150 ng/mL recombinant NRN1 protein (Abcam, ab69755) or vehicle treated with diH2O for 6 hours. NRN1 concentration was chosen based on published data that identified a plateau in exogenous NRN1 induced effects on transient potassium currents at 150 ng/mL (Yao et al., 2012). After 6 hours neurons were washed 2x with 1 mL 1X phosphate-buffered saline (PBS). To harvest cells, 1 mL 1X PBS + protease inhibitor (Fisher Scientific, catalog no. 78426) was added and cells were centrifuged for 2300rpm for 5 minutes at 4°C. Cell pellets were lysed in 200uL 8M urea buffer and HALT protease and phosphatase inhibitor cocktail (1x final concentration). Lysates were sonicated with a probe sonicator 3 times for 10 s with 10 s intervals at 30% amplitude and cleared of cellular debris by centrifugation in a tabletop centrifuge at 18,000 rcf for 3 minutes at 4° C. Protein concentration was determined by BCA assay and one-dimensional SDS-PAGE gels were run followed by Coomassie blue staining as quality control for protein integrity and equal loading before proceeding to protein digestion. Protein homogenates (50 µg) were diluted with 50 mM NH4HCO3 to a final concentration of less than 2 M urea and then treated with 1 mM dithiothreitol (DTT) at 25°C for 30 minutes, followed by 5 mM iodoacetimide (IAA) at 25°C for 30 minutes in the dark. Protein was digested with 1:100 (w/w) lysyl endopeptidase (Wako) at 25°C for 2 hours and further digested overnight with 1:50 (w/w) trypsin (Pierce) at 25°C. Resulting peptides were desalted with a Sep-Pak C18 column (Waters) and dried under vacuum.

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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)

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