scholarly journals Mouse P1 Kidney Cold-Active Protease Single Cell Dissociation v2 (protocols.io.q7ddzi6)

protocols.io ◽  
2018 ◽  
Author(s):  
Andrew Potter ◽  
Steve Potter
Keyword(s):  
Single Cell ◽  
Cell Dissociation ◽  
Cold Active ◽  
Active Protease

scholarly journals Dissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Ciara H. O’Flanagan ◽  
◽  
Kieran R. Campbell ◽  
Allen W. Zhang ◽  
Farhia Kabeer ◽  
...  
Keyword(s):  
Stress Response ◽  
Single Cell ◽  
Solid Tumor ◽  
Stress Responses ◽  
Immune Recognition ◽  
Cell Type ◽  
Collagenase Digestion ◽  
Tumor Tissues ◽  
Cold Active ◽  
Active Protease

Abstract Background Single-cell RNA sequencing (scRNA-seq) is a powerful tool for studying complex biological systems, such as tumor heterogeneity and tissue microenvironments. However, the sources of technical and biological variation in primary solid tumor tissues and patient-derived mouse xenografts for scRNA-seq are not well understood. Results We use low temperature (6 °C) protease and collagenase (37 °C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNA-seq dataset comprising 155,165 cells from patient cancer tissues, patient-derived breast cancer xenografts, and cancer cell lines. We observe substantial variation in standard quality control metrics of cell viability across conditions and tissues. From the contrast between tissue protease dissociation at 37 °C or 6 °C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, including FOS and JUN, induced by collagenase (37 °C), which are minimized by dissociation with a cold active protease (6 °C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues. Conclusions The method and conditions of tumor dissociation influence cell yield and transcriptome state and are both tissue- and cell-type dependent. Interpretation of stress pathway expression differences in cancer single-cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with the identification of such effects in dissociated scRNA-seq experiments.

scholarly journals Dissociation of solid tumour tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses

2019 ◽  
Author(s):  
Ciara H O’Flanagan ◽  
Kieran R Campbell ◽  
Allen W Zhang ◽  
Farhia Kabeer ◽  
Jamie LP Lim ◽  
...  
Keyword(s):  
Stress Response ◽  
Cell Viability ◽  
Single Cell ◽  
Mhc Class I ◽  
Cancer Cell ◽  
Solid Tumour ◽  
Cell Types ◽  
Cell Type ◽  
Cold Active ◽  
Active Protease

AbstractBackgroundSingle-cell RNA sequencing (scRNAseq) is a powerful tool for studying complex biological systems, such as tumour heterogeneity and tissue microenvironments. However, the sources of technical and biological variation in primary solid tumour tissues and patient-derived mouse xenografts for scRNAseq, are not well understood. Here, we used low temperature (6°C) protease and collagenase (37°C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNAseq dataset comprising 128,481 cells from patient cancer tissues, patient-derived breast cancer xenografts and cancer cell lines.ResultsWe observe substantial variation in standard quality control (QC) metrics of cell viability across conditions and tissues. From FACS sorted populations gated for cell viability, we identify a sub-population of dead cells that would pass standard data filtering practices, and quantify the extent to which their transcriptomes differ from live cells. We identify a further subpopulation of transcriptomically “dying” cells that exhibit up-regulation of MHC class I transcripts, in contrast with live and fully dead cells. From the contrast between tissue protease dissociation at 37°C or 6°C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, including FOS and JUN, induced by collagenase (37°C), which are minimized by dissociation with a cold active protease (6°C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues. We observe that the yield of cancer and non-cancer cell types varies between tissues and dissociation methods.ConclusionsThe method and conditions of tumour dissociation influence cell yield and transcriptome state and are both tissue and cell type dependent. Interpretation of stress pathway expression differences in cancer single cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with identification of such effects in dissociated scRNA-seq experiments.

scholarly journals Isolation and characterization of a new cold-active protease from psychrotrophic bacteria of Western Himalayan glacial soil

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Saleem Farooq ◽  
Ruqeya Nazir ◽  
Shabir Ahmad Ganai ◽  
Bashir Ahmad Ganai
Keyword(s):  
Specific Activity ◽  
Temperature Range ◽  
Psychrotrophic Bacteria ◽  
Isolation And Characterization ◽  
Cold Active ◽  
Active Protease ◽  
Quantitative Screening ◽  
First Time ◽  
Low K

AbstractAs an approach to the exploration of cold-active enzymes, in this study, we isolated a cold-active protease produced by psychrotrophic bacteria from glacial soils of Thajwas Glacier, Himalayas. The isolated strain BO1, identified as Bacillus pumilus, grew well within a temperature range of 4–30 °C. After its qualitative and quantitative screening, the cold-active protease (Apr-BO1) was purified. The Apr-BO1 had a molecular mass of 38 kDa and showed maximum (37.02 U/mg) specific activity at 20 °C, with casein as substrate. It was stable and active between the temperature range of 5–35 °C and pH 6.0–12.0, with an optimum temperature of 20 °C at pH 9.0. The Apr-BO1 had low Km value of 1.0 mg/ml and Vmax 10.0 µmol/ml/min. Moreover, it displayed better tolerance to organic solvents, surfactants, metal ions and reducing agents than most alkaline proteases. The results exhibited that it effectively removed the stains even in a cold wash and could be considered a decent detergent additive. Furthermore, through protein modelling, the structure of this protease was generated from template, subtilisin E of Bacillus subtilis (PDB ID: 3WHI), and different methods checked its quality. For the first time, this study reported the protein sequence for psychrotrophic Apr-BO1 and brought forth its novelty among other cold-active proteases.

Purification and characterization of a cold-active protease from psychrotrophicSerratia marcescensAP3801

1997 ◽  
Vol 74 (11) ◽  
pp. 1377-1383 ◽  
Author(s):  
Yasutaka Morita ◽  
Kenji Kondoh ◽  
Quamrul Hasan ◽  
Toshifumi Sakaguchi ◽  
Yuji Murakami ◽  
...  
Keyword(s):  
Purification And Characterization ◽  
Cold Active ◽  
Active Protease
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