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.

Background Single-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 sur-2 face 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.

Sunflower (Helianthus annuus L.) is a globally important oilseed, food, and ornamental crop, the second largest hybrid crop and the fourth largest oilseed crop with an estimated production value of US $20 billion (FAO, 2016). Successive rounds of artificial selection have resulted in a smaller number of alleles or less genetic diversity in cultivated sunflower than in the species as a whole (Seiler, 1992; Mandel et al., 2011), a phenomenon known as a "domestication bottleneck" (Tanksley and McCouch, 1997). However, the crosscompatible nature of sunflower allows many beneficial traits to be introgressed from wild crop relatives (WCR) into cultivated sunflower (Thompson et al., 1981; Seiler, 1992; Dempewolf et al., 2017; Seiler et al., 2017). Therefore, maintaining WCR collections and understanding their diversity are critical for sustainable sunflower production and food security.