Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of human HEK-293T cells treated with a nutraceutical formula (solution-3), or vehicle-(saline solution)-treated controls

ABSTRACT: HEK-293T cells were plated (1x106 cells) and treated with a nutraceutical formula (solution-3) at 0.01x concentration or with vehicle (saline solution) as the negative control for the treatment. After 24 hours, the cells were lysed, and their RNA was extracted for RNAseq.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: Massimo Zollo

PROVIDER: E-MTAB-13918 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:CHD5 is frequently deleted in neuroblastoma, and appears to be a tumor suppressor gene; however, little is known about the role of CHD5. We found CHD5 mRNA was restricted to brain; by contrast most other remodeling ATPases were broadly expressed. CHD5 protein isolated from mouse brain was associated with HDAC2, p66, MTA3 and RbAp46 in a megadalton complex. CHD5 protein was detected in several rat brain regions and appeared to be enriched in neurons. CHD5 protein was predominantly nuclear in primary rat neurons and brain sections. Microarray analysis revealed genes that were upregulated and downregulated when CHD5 was depleted from primary neurons. CHD5 depletion altered expression of neuronal genes, transcription factors, and brain-specific subunits of the SWI/SNF remodeling enzyme. Aging and Alzheimers gene sets were strongly affected by CHD5 depletion from primary neurons. Chromatin immunoprecipitation revealed CHD5 bound to these genes, suggesting the regulation was direct. Together, these results indicate that CHD5 is found in a NuRD-like multi-protein complex. CHD5 is restricted to the brain, unlike the closely related family members CHD3 and CHD4. CHD5 regulates expression of neuronal genes, cell cycle genes and remodeling genes. CHD5 is linked to regulation of aging and Alzheimer’s genes. CHD5 KD shRNA sequences were designed according to the instructions for the pLKO.1 system (Addgene). Control was as described (Sci307-1098,2005) Scramble, clone 1864 from Addgene). Virus was packaged using HEK-293T cells, pLKO.1 vector with shRNA inserts for CHD5, and the control. 48 hours after transfection of 293 cells, medium containing virus was filtered (0.45 micron), then applied for 6 hours to primary cortical neurons one day after the neurons were plated (Day 1). Medium was removed, and replaced with Neural Basal Medium, and the cells were cultured until Day 5, 9 or 12. RNA was harvested from 3 replicates of the treated primary cortical neurons at each time point. RNA was isolated using RNAeasy Kit (Qiagen), Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips. RNA was labeled using the standard Illumina protocol and Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) Biotin labeled cRNA was hybridized to Illumina's Sentrix Rat Ref-12 v1 Expression BeadChips.

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RNA-seq of human HEK-293T cells treated with a nutraceutical formula (solution-3), or vehicle-(saline solution)-treated controls

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