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The use of cold active proteases can dramatically reduce single cell RNA-seq gene expression artifacts

ABSTRACT: Single cell RNA-seq is a powerful methodology, but with important limitations. In particular, the process of enzymatic separation of cells at 37O C can be expected to result in artifact changes in gene expression patterns. We here describe a dissociation method that uses protease from a psychrophilic microorganism with high activity in the cold. The entire procedure is carried out at 6O C or colder, where mammalian transcriptional machinery is largely inactive. To test this method we carry out single cell RNA-seq on about 9,000 cells, comparing the results of the cold method with a method using 37O C incubations for multiple times. We show that the cold active protease method results in a great reduction in gene expression artifacts.

ORGANISM(S): Mus musculus

PROVIDER: GSE94333 | GEO | 2017/02/01

SECONDARY ACCESSION(S): PRJNA369393

REPOSITORIES: GEO

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Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: a molecular atlas of kidney development.

Project description:Single-cell RNA-seq is a powerful technique. Nevertheless, there are important limitations, including the technical challenges of breaking down an organ or tissue into a single-cell suspension. Invariably, this has required enzymatic incubation at 37°C, which can be expected to result in artifactual changes in gene expression patterns. Here, we describe a dissociation method that uses a protease with high activity in the cold, purified from a psychrophilic microorganism. The entire procedure is carried out at 6°C or colder, at which temperature mammalian transcriptional machinery is largely inactive, thereby effectively 'freezing in' the in vivo gene expression patterns. To test this method, we carried out RNA-seq on 20,424 single cells from postnatal day 1 mouse kidneys, comparing the results of the psychrophilic protease method with procedures using 37°C incubation. We show that the cold protease method provides a great reduction in gene expression artifacts. In addition, the results produce a single-cell resolution gene expression atlas of the newborn mouse kidney, an interesting time in development when mature nephrons are present yet nephrogenesis remains extremely active.

| S-EPMC5665481 | biostudies-literature

Transcriptome of psychrophilic fungus Mrakia psychrophila cultured at different temperature at 4,12,20 degree centigrade

Project description:Mrakia psychrophlila is a psychrophilic fungus widely spread all over the world. The cold adaptation mechanism of Mrakia psychrophila is unknown. Using RNA sequencing, differentially expressed genes of Mrakia psychrophila cultured at 4, 12 and 20 degree centigrade are identified. The result may be benefit for understanding cold adaptation of microorganisms.

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Cold adaptation of tRNA nucleotidyltransferases: a tradeoff in activity, stability and fidelity

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Supercooled, subzero, high salinity incubations of Cp34H reveal long-term mitigation strategies and detectable (peptide) biomarkers for life on other icy worlds

Project description:Colwellia psychrerythraea is a marine psychrophilic bacterium known for its remarkable ability to maintain activity during long-term exposure to extreme subzero temperatures and correspondingly high salinities in sea ice. These microorganisms must have simultaneous adaptations to high salinity and low temperature to survive, be metabolically active, or grow in the ice. Here we report results obtained through an experimental design that allowed us to monitor culturability, activity, and proteomic signatures of Colwellia psychrerythraea strain 34H (Cp34H) to subzero temperature, salinity, and nutrient availability by performing long-term incubations in eight different conditions. Shotgun proteomics revealed novel metabolic strategies used to maintain culturability in response to each independent experimental variable, particularly in pathways regulating carbon, nitrogen, and fatty acid metabolism. For the first time, statistical analysis of differential abundances of proteins uniquely identified in these isolated conditions provide metabolism-specific protein biosignatures indicative of growth or survival in either increased salinity, decreased temperature, or nutrient limitation. Additionally, to aid in the search for extant life on other icy worlds, analysis of detected short peptides enriched and retained in -10oC incubations for four months identified over 400 potential biosignatures that could indicate the presence of terrestrial-like cold-active or halophilic metabolisms on other icy worlds.

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Peptide gene sequences of substrate phage for different proteases

Project description:We present a method that employs two genetically encoded substrate phage display libraries coupled with next generation sequencing (SPD-NGS) that allows up to 10,000-fold deeper sequence coverage of the typical 6 to 8 residue protease cleavage sites compared to state-of-the-art synthetic peptide libraries or proteomics. We applied SPD-NGS to two classes of proteases, the intracellular caspases 2, 3, 6, 7 and 8, and the ectodomains of the membrane sheddases, ADAMs 10 and 17. The first library (Lib 10AA) was used to determine substrate cleavage motifs. Lib 10AA contains a highly diverse randomized 10-mer substrate peptide sequences (10^9 unique members) that was displayed mono-valently on filamentous phage and bound to magnetic beads via an N-terminal biotin. The protease was allowed to cleave the SPD beads, and the released phage subjected to up to three total rounds of positive selection followed by next generation sequencing (NGS). This allowed us to identify from 10^4 to 10^5 unique cleavage sites over a broad dynamic range of NGS counts (ranging from 3-5000), and produced consensus and optimal cleavage motifs based positional sequencing scoring matrices and state-of the-art machine learning algorithm that closely matched synthetic peptide data. A second SPD-NGS library (Lib hP) was constructed that allowed us to identify candidate human proteome sequences. Lib hP displayed virtually the entire human proteome tiled in contiguous 49AA sequences with 25AA overlaps (nearly 1 million members). After three rounds of positive selection we identified up to 10^4 natural linear cut sites depending on the protease and captured most of the examples previously identified by proteomics (ranging from 30 to 1000) and predicted 10 to 100-fold more.

2020-07-23 | GSE154923 | GEO

Microarray gene expression profiles from temperature acclimated atrial, ventricular compact and spongy tissues of Pacific bluefin tuna Thunnus orientalis

Project description:In this study, we examined the transcriptomic responses to temperature acclimation (14oC, 20oC, and 25oC) in atrial and ventricular tissues of Pacific bluefin tuna (PBFT). A global gene expression analysis using a bluefin tuna-specific microarray indicated profound changes in expression of genes associated with energy metabolism, protein turnover, cellular stress response, oxidative stress and apoptosis. A principal component analysis revealed tissue-specific transcriptomic responses to temperature, with atrium at 25oC showing the greatest variation. Overall transcriptomic data suggests that PBFT can optimize cardiac function in the cold by acclimating to 14oC. Capacity to acclimate to colder temperatures potentially underlies this species ability to expand its vertical and horizontal thermal niche and migrate to colder oceans at high latitudes. In contrast, the cardiac phenotype of 25oC acclimated fish infers that PBFT hearts struggle to maintain cellular homeostasis and are subjected to programmed cell death. The goal of this study was to compare transcriptomic response to cold and warm temperature acclimations across cardiac tissues in Pacific bluefin tuna. Fish (n=4) were acclimated to 14C, 20C and 25C, and RNA was extracted from atrial, ventricular compact and spongy tissues. Experimental samples were hybridyzed against a reference pool that contained a mix of RNA from every sample.

2012-03-31 | E-GEOD-36886 | biostudies-arrayexpress

Proteomics of Colwellia psychrerythraea grown at multiple subzero temperatures

Project description:The mechanisms that allow psychrophilic bacteria to remain metabolically active at subzero temperatures result from form and function of their proteins. We present first proteomic evidence of physiological changes of the marine psychrophile Colwellia psychrerythraea 34H (Cp34H) after exposure to subzero temperatures (-1, and -10°C in ice) through 8 weeks. Protein abundance was compared between different treatments to understand the effects of temperature and time, independently and jointly, within cells transitioning to, and being maintained in ice. Parallel [3H]-leucine and [3H]-thymidine incubations indicated active protein and DNA synthesis to -10°C. Mass spectrometry-based proteomics identified 1763 proteins across four experimental treatments. Proteins involved in osmolyte regulation and polymer secretion were found constitutively present across all treatments, suggesting that they are required for metabolic success below 0°C. Differentially abundant protein groups indicated a reallocation of resources from DNA binding to DNA repair and from motility to chemo-taxis and sensing. Changes to iron and nitrogen metabolism, cellular membrane structures, and protein synthesis and folding were also revealed. By elucidating vital strategies during life in ice, this study provides novel insight into the extensive molecular adaptations that occur in cold-adapted marine organisms to sustain cellular function in their habitat. Raw data available at http://www.chorusproject.org Public project ID number 682.

2019-02-15 | PXD006274 | Pride

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Project description:The RNA-Seq analysed the changes in protease gene expression upon short-term PI3K inhibition in three breast cancer cell lines.

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Microarray gene expression profiles from temperature acclimated atrial, ventricular compact and spongy tissues of Pacific bluefin tuna Thunnus orientalis

2012-04-01 | GSE36886 | GEO

Rat Astrocyte-Neuron Co-cultures versus purified co-cultured Astrocytes versus Astrocyte alone

Project description:Profiling of transcriptional changes in rat astrocytes when co-cultured with neurons: comparison of astrocytes cultured alone with astrocytes co-cultured with mouse hippocampal neurons. Co-cultured astrocytes are isolated using cold jet, a novel tool for these neuron-glia cultures. Over the last decade, the importance of astrocyte-neuron communication in neuronal development and synaptic plasticity has become increasingly clear. Since neuron-astrocyte interactions represent highly dynamic and reciprocal processes, we hypothesized that at least part of the involved astrocyte genes may be regulated as a consequence of their interactions with maturing neurons. In order to identify such neuron-induced astrocyte genes in vitro, we tested the effectiveness of the ‘cold jet’, a new method for separation of neurons from co-cultured astrocytes. The cold jet method is performed under ice-cold conditions and avoids protease-mediated isolation of astrocytes or time-consuming centrifugation, yielding intact astrocyte mRNA with approximately 90% of neuronal RNA removed. Using this method, we executed genome-wide profiling in which RNA derived from astrocyte-only cultures was compared with astrocyte RNA derived from differentiating neuron-astrocyte co-cultures. Data analysis revealed changes in expression of a large number of mRNAs and biological processes, including novel findings. Thus, cold jet is an efficient method to separate astrocytes from neurons in co-culture, and in this study reveals that neurons induce robust gene-expression changes in co-cultured astrocytes.

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