Enzymatic dissociation induces transcriptional and proteotype bias in brain cell populations
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ABSTRACT:
PROVIDER: PRJNA601729 | ENA |
REPOSITORIES: ENA
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Project description: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.
2021-09-08 | PXD015592 | Pride
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
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
Project description:The use of single cell transcriptomics provides previously inaccessible insights into cellular heterogeneity and lineage dynamics of the mammary gland allowing for a better understanding of normal mammary gland function as well as breast cancer initiation and progression. Especially for human mammary gland research, limited tissue accessibility and restriction to ex vivo techniques reinforce the importance of reliable cross-study comparison of single-cell transcriptomic data. However, it is unclear to what extent differences in breast tissue dissociation influence composition and transcriptomic profiles of isolated cells. Here, we used single-cell RNA sequencing to compare human mammary cell populations isolated from a single mammoplasty patient by varying enzymatic dissociation protocols differing in duration (3 or 16 hours) and agitation speed (10 rpm or 100 rpm). Protocol A (3 hours, 100 rpm), protocol B (16 hours, 100 rpm) and protocol C (16 hours, 10 rpm) were used to extract cell fragments from tissue which were either frozen down directly or further dissociated into single cells prior to cryopreservation. Samples were then prepared together for 10x scRNA-sequencing, where the fragments were defrosted and freshly dissociated and were loaded together with the defrosted single cells. From each of the protocols we sequenced similar numbers of cells isolated from fragments (Protocol A: 3,586 cells, Protocol B: 2,809 cells and Protocol C: 4,796 cells), finding an average of 7,427 unique molecular identifiers and 2,445 genes detected per cell. Overall, we detected a greater abundance and heterogeneity of stromal cell types, such as fibroblasts and endothelial cells at a lower agitation speed. Moreover, an extended duration of tissue dissociation governed an overall cellular oxidative stress response together with a downregulation of breast cancer associated genes and a cell-type specific downregulation of lineage markers. Thus, our systematic analysis of dissociation-induced compositional and transcriptional bias in human breast tissue samples yields useful information to avoid misinterpretation of cellular heterogeneity and lineage composition.
2023-06-30 | E-MTAB-9844 | biostudies-arrayexpress
Project description: Not available
| S-EPMC7663484 | biostudies-literature
Project description:Shp-deletion induces dissociation of steatosis and inflammation in NASH
| PRJNA551941 | ENA
Project description:Tissue dissociation, mechanical and/or enzymatic, is a method of disaggregating 3D connective tissue to release cells for downstream single cell analysis. Without proper controls for tissue dissociation experiments, potential artifacts can be mistaken for baseline tissue data. We hypothesized that enzymatic (collagenase/hyaluronidase) compared to mechanical dissociation leads to dissociated related artifacts not found in endogenous tumor tissue. To distinguish artifacts between enzymatic and mechanical dissociation, we compared separate enzymatic and mechanical dissociated samples with baseline samples from healthy and tumor patient whole blood, melanoma and head and neck squamous cell carcinoma (HNSCC) tumor tissues in flow cytometry, RNA sequencing and CODEX experiments. In bulk sorted tumor tissue, there is a significant difference between enzymatic and mechanical digested samples in cell stress, heat shock proteins and cell death related genes. These genes were also significantly upregulated in enzymatic over mechanical samples in the single cell RNA sequencing HNSCC tissue data. Fibroblasts and CD39+CD103+ CD8 T cells from tumor tissue, appear to require enzymatic digestion for efficient release from tissue compared to the mechanical digested samples. CD39+CD103+ CD8 tumor infiltrating lymphocytes (TILs) show different phenotypes depending on dissociation technique and tumor tissue. In tumor tissue, CD3+CD8+CD69+ T cells and gene expression were elevated in the enzymatic samples over the mechanical and baseline frozen tissue. Tissue dissociation techniques and tissue analysis from downstream applications have the potential for dissociation related artifacts that can mislead both protein and RNA sequencing data analysis in cancer research.
2023-09-19 | GSE243360 | GEO
Project description:Tissue dissociation, mechanical and/or enzymatic, is a method of disaggregating 3D connective tissue to release cells for downstream single cell analysis. Without proper controls for tissue dissociation experiments, potential artifacts can be mistaken for baseline tissue data. We hypothesized that enzymatic (collagenase/hyaluronidase) compared to mechanical dissociation leads to dissociated related artifacts not found in endogenous tumor tissue. To distinguish artifacts between enzymatic and mechanical dissociation, we compared separate enzymatic and mechanical dissociated samples with baseline samples from healthy and tumor patient whole blood, melanoma and head and neck squamous cell carcinoma (HNSCC) tumor tissues in flow cytometry, RNA sequencing and CODEX experiments. In bulk sorted tumor tissue, there is a significant difference between enzymatic and mechanical digested samples in cell stress, heat shock proteins and cell death related genes. These genes were also significantly upregulated in enzymatic over mechanical samples in the single cell RNA sequencing HNSCC tissue data. Fibroblasts and CD39+CD103+ CD8 T cells from tumor tissue, appear to require enzymatic digestion for efficient release from tissue compared to the mechanical digested samples. CD39+CD103+ CD8 tumor infiltrating lymphocytes (TILs) show different phenotypes depending on dissociation technique and tumor tissue. In tumor tissue, CD3+CD8+CD69+ T cells and gene expression were elevated in the enzymatic samples over the mechanical and baseline frozen tissue. Tissue dissociation techniques and tissue analysis from downstream applications have the potential for dissociation related artifacts that can mislead both protein and RNA sequencing data analysis in cancer research.
2023-09-19 | GSE243359 | GEO
Project description:ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics
| PRJNA631686 | ENA
Project description:ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics
| PRJNA702503 | ENA