![]() While current technologies attempt to mitigate noise from amplification during library construction by the incorporation of unique molecular identifiers (UMIs) during cDNA synthesis, this does not address changes to the transcriptome prior to reverse transcription. Inherent variations in tissue composition, cell quality, and cell-cell variability can also make it difficult to confidently interpret scRNA-seq data. In addition, the transcriptional behavior of single cells can deviate profoundly from the population as a whole, and gene expression pulse patterns have been shown to contribute significant noise levels to scRNA-seq data. The inherent nature of the transcriptome is transient and dynamic, reflecting the ability of cells to quickly respond to their environment. scRNA-seq data is also subject to technical and biological noise. Due to the sensitivity of single-cell RNA sequencing (scRNA-seq), small changes in gene expression can dramatically influence the interpretation of biological data. Recent advancements in sequencing technologies have allowed for RNA sequencing at single-cell resolution, which can be used to interrogate features of tumor tissues that may not be resolved by bulk sequencing, such as intratumoral heterogeneity, microenvironmental architecture, clonal dynamics, and the mapping of known and de novo cell types. We define a core set of 512 genes that can assist with the identification of such effects in dissociated scRNA-seq experiments. 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. The method and conditions of tumor dissociation influence cell yield and transcriptome state and are both tissue- and cell-type dependent. 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 derive a core gene set of 512 heat shock and stress response genes, including FOS and JUN, induced by collagenase (37 ☌), which are minimized by dissociation with a cold active protease (6 ☌). From the contrast between tissue protease dissociation at 37 ☌ or 6 ☌, we observe that collagenase digestion results in a stress response. We observe substantial variation in standard quality control metrics of cell viability across conditions and tissues. We use low temperature (6 ☌) protease and collagenase (37 ☌) 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. 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. TrypLE products offer a greener, more economical cell dissociation.Single-cell RNA sequencing (scRNA-seq) is a powerful tool for studying complex biological systems, such as tumor heterogeneity and tissue microenvironments. ![]() In addition, TrypLE enzymes boast similar dissociation kinetics to porcine trypsin as well as comparable cell replating, proliferation kinetics, and long-term maintenance. Instead, it is inactivated by dilution alone. So gentle, in fact, that the TrypLE enzyme requires no inhibiting agent. TrypLE enzymes can be substituted for trypsin in existing protocols, is room temperature stable and, in addition, is exceedingly gentle on cells. The TrypLE enzyme is designed to seamlessly fit your workflow. With the TrypLE enzyme’s animal-free origin, hazards from potential pathogenic contaminants have been eliminated and subject to a controlled fermentation process, TrypLE enzymes are available for supply at any scale. Exhibiting a similar pH activity profile to trypsin, this enzyme cleaves at arginine and lysine. Ideal for both serum-supplemented and serum-free conditions, TrypLE enzymes are an answer to animal origin-free trypsin-like enzymes. Gibco TrypLE gentle cell dissociation reagents
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