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Protrusion vs cell-body mRNA analysis by RNA-seq

ABSTRACT: We quantified the distribution of cellular mRNAs between protrusions and cell-body of MDA-MB-231 breast carcinoma cells by RNA-seq, using a filter based fractionation method. In this method, cells are seeded on top of 3micrometer transwell filters to enable protrusions to form but to prevent the cell-bodies passing through due to the small size of the pores, thus resulting in separation of protrusions and cell-bodies on opposite sides of the filter, from which RNA can be prepared separately. RNA samples for Illumina sequencing were generated in duplicates from two biological replicate experiments (two protrusions and two cell-body).

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Homo sapiens

SUBMITTER: Faraz Mardakheh

PROVIDER: E-MTAB-2546 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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RNA-seq of coding RNAs from protrusions of MDA-MB231 cells treated with control or exosome core complex siRNA

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Combining transcriptome and proteome analysis to identify and characterize the RpoS regulon in Pseudomonas fluorescens

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2019-11-12 | PXD010845 | Pride

Protrusion vs Cell-body SILAC proteomics

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Quantitative proteomics analysis of actin-rich protrusions and cell-bodies in migratory cells

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2020-10-20 | PXD021239 | Pride

H2S regulation the metabolism of cucumber in response to salt-stress through transcriptome and proteome analysis

Project description:In a previous study, we found that H2S alleviates salinity stress in cucumber by maintaining the Na+/K+ balance and by regulating H2S metabolism and the oxidative stress response. However, little is known about the molecular mechanisms behind H2S-regulated salt-stress tolerance in cucumber. Here, an integrated transcriptomic and proteomic analysis based on RNA-seq and 2-DE was used to investigate the global mechanism underlying H2S-regulated salt-stress tolerance. In total, 11 761 differentially expressed genes (DEGs) and 61 differentially expressed proteins (DEPs) were identified. Analysis of the pathways associated with the DEGs showed that salt stress enriched expression of genes in primary and energy metabolism, such as photosynthesis, carbon metabolism and biosynthesis of amino acids. Application of H2S significantly decreased these DEGs but enriched DEGs related to plant-pathogen interaction, sulfur-containing metabolism, cell defense and signal transduction pathways. Notably, changes related to sulfur-containing metabolism and cell defense were also observed through proteome analysis, such as Cysteine synthase 1, Glutathione S-transferase U25-like, Protein disulfide-isomerase and Peroxidase 2. We present the first global analysis of the mechanism underlying H2S regulation of salt-stress tolerance in cucumber through tracking changes in the expression of specific proteins and genes.

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Project description:In mesenchymal-like cell migration, cells need to polarise into a protrusive front and a retracting cell body. Apart from proteins, several mRNAs are known to be localised to cell protrusions but to what extent RNA localisation and local translation contributes toward front-back assymetry is unknown. We set out to quantify the relative translation rates of cellular proteins between protrusions and cell-body by Pulsed-SILAC proteomics, using MDA-MB-231 cells, a highly invasive breast cancer cell-line. We utilised a transwell filter based fractionation method in conjugation with pulsed-SILAC. In this method, cells are seeded on top of 3μm transwell filters to enable protrusions to form through the pores of the filters but to prevent the cell-bodies passing through due to the small size of the pores, thus resulting in separation of protrusions and cell-bodies on opposite sides of the filter, which can then be lysed and prepared separately. Prepration of protrusion and cell-body fractions from SILAC heavy vs. medium label pulsed cells then allows for reciprocal mixing and quantification of nascent proteins between protrusions and cell-body. We determined the relative local translation rates of 1150 proteins between protrusions and cell-body from two pulsed-SILAC mixes.

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QUANTSEQ 3' sequencing of protrusions and cell bodies of control (NT) or LARP6 siRNA (LARP6 KD) transfected MDA-MB231 breast cancer cells

Project description:To assess how LARP6 affects mRNA localisation to cell-protrusions, we transfected MDA-MB231 cells with either non-targeting control siRNA or LARP6 siRNA for 72 hrs, before fractionating the cells into protrusion and cell-bodies, and analysed total RNA from each fraction via Lexogen QUANTSEQ FWD 3' mRNA sequencing. Protrusion induction was done for 2 hrs on 3 micron transwell filters (Corning). The experiment was performed twice using two independent batches of cells, and sequencing was carried out on an Illumina NextSeq 500 sequencer.

2020-10-30 | E-MTAB-9520 | biostudies-arrayexpress

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