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Enzymatic dissociation induces transcriptional and proteotype bias in brain cell populations

ABSTRACT: Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here we show that standard enzymatic digestion of brain tissue at 37°C induces profound alterations in the transcriptome and proteotype of specific brain cells, as compared to cold mechanical dissociation. These findings emphasize the need to consider intrinsic bias and biological artefacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.

INSTRUMENT(S): Orbitrap Fusion Lumos, Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain

SUBMITTER: Marc van Oostrum

LAB HEAD: Bernd Wollscheid

PROVIDER: PXD015592 | Pride | 2021-09-08

REPOSITORIES: Pride

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	Astro-DDA-1.raw	Raw
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	Astro-DDA-3.raw	Raw
	Astro-DDA-4.raw	Raw
	Astro-DIA-37-1.raw	Raw

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Publications

Enzymatic Dissociation Induces Transcriptional and Proteotype Bias in Brain Cell Populations.

Mattei Daniele D Ivanov Andranik A van Oostrum Marc M Pantelyushin Stanislav S Richetto Juliet J Mueller Flavia F Beffinger Michal M Schellhammer Linda L Vom Berg Johannes J Wollscheid Bernd B Beule Dieter D Paolicelli Rosa Chiara RC Meyer Urs U

International journal of molecular sciences 20201026 21

Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here, we provide a systematic investigation of the influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia. We show that standard enzymatic digestion of brain tissue at 37 °C induces profound ...[more]

PMID: 33114694

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Enzymatic dissociation induces transcriptional and proteotype bias in brain cell populations

Project description:Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here, we provide a systematic investigation of the influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia. We show that standard enzymatic digestion of brain tissue at 37°C induces profound and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared to an optimized mechanical dissociation protocol at 4°C. These findings emphasize the risk of introducing technical biases and biological artefacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.

2020-10-26 | GSE143796 | GEO

Enzymatic dissociation induces transcriptional and proteotype bias in brain cell populations

Project description:Enzymatic dissociation induces transcriptional and proteotype bias in brain cell populations

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Universal correction of enzymatic sequence bias

Project description:Coupling molecular biology to high throughput sequencing has revolutionized the study of biology. Molecular genomics techniques are continually refined to provide higher resolution mapping of nucleic acid interactions and nucleic acid structure. These assays are converging on single-nucleotide resolution measurements, but the sequence preferences of molecular biology enzymes can interfere with the accurate interpretation of the data. Enzymatic sequence preferences manifest more prominently as the resolution of these assays increase. We developed seqOutBias to seek out enzymatic sequence bias from experimental data and scale individual sequence reads to correct the bias. We show that this software efficiently and successfully corrects the sequence bias resulting from DNase-seq, TACh-seq, ATAC-seq, MNase-seq, and PRO-seq data.

2016-12-21 | GSE92674 | GEO

Identification of quiescence and early activation genes in satellite cells

Project description:We report the transcriptional changes that occur during the isolation procedure of adult skeletal muscle stem cells (MuSCs). We have developed a protocol that is based on the in situ fixation of MuSCs before isolation. The transcriptome of the in situ fixed cells was compared to that of MuSCs isolated using the standard mechanical/enzymatic isolation protocol. The differentially expressed transcripts represent early-response genes, involved in quiescence and activation of MuSCs, but also isolation-induced artefacts.

2017-11-13 | GSE101576 | GEO

De novo Classification of Mouse B Cell Types using Surfaceome Proteotype Maps

Project description:System-wide quantification of the cell surface proteotype and identification of extracellular glycosylation sites is challenging when sample is limiting. We miniaturized and automated the previously described Cell Surface Capture technology increasing sensitivity, reproducibility, and throughput. We used this technology, which we call autoCSC, to create population-specific surfaceome maps of developing mouse B cells and used targeted flow cytometry to uncover developmental cell subpopulations.

2019-12-11 | PXD013627 | Pride

Efficient enzyme-free isolation of brain-derived extracellular vesicles

Project description:Extracellular vesicles (EVs) have gained significant attention as potential diagnostic and therapeutic tools due to their role in intercellular communication and their ability to encapsulate various biomolecules. However, the isolation of EVs from tissue remains challenging, often requiring enzymatic digestion steps that may introduce unwanted biases and affect EV integrity. In this study, we present an efficient non-enzymatic approach for the isolation of brain-derived extracellular vesicles (BDEVs). Our method employs sequential ultracentrifugation and a density-based gradient to achieve a high yield and purity of BDEVs from brain tissue without any enzymatic digestion. Characterization of the isolated EVs revealed their typical morphology and size distribution, as confirmed by transmission electron microscopy and nanoparticle tracking analysis. We show by western blot that known BDEV markers, such as Flotlin-1, CD81, Alix, and the prion protein (PrP) are present without proteolytic cuts introduced by the enzymatic digestion of the tissue. We also show that the absence ofGM130 ,widely accepted as a negative marker to assess BDEV’s purity, is due to enzymatic digestion rather than lack of this protein in the sample. Moreover, we find no differences in mRNA content between the non-enzymatically and the enzymatically isolated BDEVs, showing that the intraluminal part of the BDEVs is not affected by enzymatic isolation. Similarly, our proteomic analysis shows unaltered protein expression in BDEVs isolated with both methods. However, other proteins, potentially in the BDEVs corona and membrane, are differentially expressed, highlighting the consequences of the enzymatically-based isolation protocol.

2024-11-07 | PXD045737 | Pride

BCL-2 modulates IRE1a activation to attenuate ER stress and pulmonary fibrosis

Project description:BCL-2 modulates IRE1a activation to attenuate ER stress and pulmonary fibrosis. Mice were treated with Bleomycin + Vehicle or Bleomycin + Navitoclax. Lungs were harvested 21 days after bleomycin injury followed by enzymatic digestion of the lungs to create single cell suspension and flow-sorting based isolation of total epithelial and stromal cells

2024-03-04 | GSE244568 | GEO

The enzymatic activities of CD38 enhance CLL growth and trafficking: implications for therapeutic targeting

Project description:The ecto-enzyme CD38 is a marker of unfavorable prognosis for chronic lymphocytic leukemia (CLL) patients and an indicator of activation and proliferation of leukemic cells. Here we show that CD38 is enzymatically active in primary CLL cells and that its forced expression increases disease aggressiveness in a xenograft model. The effect is completely lost when using an enzyme deficient version of CD38 with a single amino-acid mutation. Through the enzymatic conversion of NAD, CD38 increases cytoplasmic Ca2+ concentrations, positively influencing proliferation, chemotaxis, adhesion and matrix digestion. Inhibition of the enzymatic activities of CD38 using the flavonoid kuromanin blocks CLL homing. In a short-term xenograft model using primary cells, kuromanin treatment traps CLL cells in the blood, increasing responses to chemotherapy.

2014-04-01 | GSE56369 | GEO

The enzymatic activities of CD38 enhance CLL growth and trafficking: implications for therapeutic targeting

2014-04-01 | E-GEOD-56369 | biostudies-arrayexpress

Characterization of an astrocyte perivascular endfeet transcriptome

Project description:We aim to identify mRNAs present in astrocyte endfeet. We performed a microarray study comparing mRNAs extracted from brain vessels purified from 2-month-old C57Bl6 mice, submitted or not to a partial basal lamina enzymatic digestion, allowing to remove astrocyte endfeet membranes and their associated astroglial mRNAs from the purified vessels. mRNAs depleted upon basal lamina digestion represent a specific population of astrocyte mRNAs transported in the perivascular endfeet

2017-06-14 | GSE76299 | GEO

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