Single cell RNA-seq profiling of T cells isolated from MC38 mouse tumors
ABSTRACT: We investigate the single-cell landscape of the inflammatory mouse tumor model MC38, a C57BL/6 tumor cell line derived from colon adenocarcinoma. MC38 (diluted in HBSS and matrigel) was inoculated in the right unilateral flank (in the border of positions B2 and B3) of C57BL/6 mice (ref Study 16-3384 AV). Tumors were taken one day after group-out (average 150-250 mm3 at day 0), approximately 14-19 days. Tissues were dissociated and flow sorted accordingly to obtain the following groups for 10x Chromium 5' Gene Expression Profiling. Our results indicate that the degree of clonal expansion is correlated with expression of T cell exhaustion markers, and that T cells with strong exhaustion phenotype also express high levels of activation markers, such as interferon gamma.
Project description:We investigate the single-cell landscape of the inflammatory mouse tumor model MC38, a C57BL/6 tumor cell line derived from colon adenocarcinoma. MC38 (diluted in HBSS and matrigel) was inoculated in the right unilateral flank (in the border of positions B2 and B3) of C57BL/6 mice (ref Study 16-3384 AV). Tumors were taken one day after group-out (average 150-250 mm3 at day 0), approximately 14-19 days. Tissues were dissociated and flow sorted accordingly to obtain the following groups for 10x Chromium 5' Gene Expression Profiling. Our results indicate that the degree of clonal expansion is correlated with expression of T cell exhaustion markers, and that T cells with strong exhaustion phenotype also express high levels of activation markers, such as interferon gamma.
Project description:We report scRNA-seq data captured from 9,410 cells obtained from the skin of K14E7 transgenic and wildtype C57/BL6 mice. The K14E7 mouse model harbors the HPV16 E7 oncogene driven from a Keratin 14 promoter for keratinocyte-specific expression. We used scRNA-seq to detect and measure E7 transcription with unprecedented accuracy and resolution. With these data, we uncovered transcriptional differences between the individual cells; demonstrated that increased HPV16 E7 copy number is associated with increased expression of E7-induced genes; and showed that E7 expression is predominantly associated with basal keratinocytes.
Project description:We characterized single-cell transcriptional profiles of the cardiac non-myocyte cell pool in C57BL/6J mice. The cell preparation we sequenced consisted of metabolically active, nucleated non-myocyte cells from heart ventricles of female and male mice which were depleted of endothelial cells. The goals of this experiment included examining cellular diversity, identifying markers of understudied cell populations, exploring functional roles of different cell types, and characterizing sexual dimorphism in cardiac gene expression.
Project description:We used the resolving power of single-cell transcriptional profiling to molecularly characterize the mouse adipose stem and progenitor cell-enriched, subcutaneous adipose stromal vascular fraction. We molecularly assessed CD45- CD31- SVF cells using the 10x Genomics Chromium (10x) platform.
Project description:We used human embryonic stem cell-derived retinal ganglion cells (RGCs) to characterize the transcriptome of 1,174 cells at the single cell level. The human embryonic stem cell line BRN3B-mCherry A81-H7 was differentiated to RGCs using a guided differentiation approach. Cells were harvested at day 36 and incubated with THY1 antibody (Miltenyi) before undergoing FACS. THY1 positive and THY1 negative cells were subsequently prepared for single cell RNA sequencing. Single cell suspensions were loaded onto 10X Genomics Single Cell 3' Chips along with the reverse transcription master mix as per the manufacturer's protocol for the Chromium Single Cell 3' v2 Library (10X Genomics; PN-120233), to generate single cell gel beads in emulsion. Libraries were then sequenced on an Illumina HiSeq 2500.
Project description:The aims of the experiment were to profile the cell types in the adult mouse cardiac interstitium (non-myocute cells) and how they respond to myocardial infarction injury. Adult, male, Pdgfra +/GFP mice were subject to either a myocardial infarction or sham injury, with cells isolated from cardiac ventricles 3 or 7 days following surgery. We obtained scRNA-seq profiles of two cell fractions: total interstitial (non-myocyte) cell population (TIP) and FACS-sorted GFP+/Cd31- cells (GFP).
Project description:Spermatogenesis is a recurring differentiation process that results in the production of male gametes within the testes. During this process, spermatogonial stem cells differentiate to form spermatocytes, which undergo two rounds of meiotic division to form haploid spermatids. Throughout spermiogenesis, round spermatids elongate to form mature sperm. To profile maturing germ cells during the first wave of spermatogenesis, we generated droplet-based single cell RNAseq from juvenile animals at post-natal day P5-P35 as well as adult animals. Cells were isolated from whole testes. Furthermore, to assay the robustness of the meiotic cell division process, we profiled germ cells of the trans-chromosomal mouse model (Tc1) that carries a copy of the human chromosome 21.
Project description:Barcode swapping results in the mislabeling of sequencing reads between multiplexed samples on the new patterned flow cell Illumina sequencing machines. This may compromise the validity of numerous genomic assays, especially for single-cell studies where many samples are routinely multiplexed together. The severity and consequences of barcode swapping for single-cell transcriptomic studies remain poorly understood. We have used two statistical approaches to robustly quantify the fraction of swapped reads in each of two plate-based single-cell RNA sequencing datasets. We found that approximately 2.5% of reads were mislabeled between samples on the HiSeq 4000 machine, which is lower than previous reports. We observed no correlation between the swapped fraction of reads and the concentration of free barcode across plates. Further- more, we have demonstrated that barcode swapping may generate complex but artefactual cell libraries in droplet-based single-cell RNA sequencing studies. To eliminate these artefacts, we have developed an algorithm to exclude individual molecules that have swapped between samples in 10X Genomics experiments, exploiting the combinatorial complexity present in the data. This permits the continued use of cutting-edge sequencing machines for droplet-based experiments while avoiding the confounding effects of barcode swapping. This data repository contains the sequencing files associated with the droplet based scRNA-seq dataset in Griffiths et al. (2018). The data presented here should purely used for technical analysis, the biological motivation is nonetheless briefly described in the following: The mammary gland is a unique organ as it undergoes most of its development during puberty and adulthood. Characterising the hierarchy of the various mammary epithelial cells and how they are regulated in response to gestation, lactation and involution is important for understanding how breast cancer develops. Recent studies have used numerous markers to enrich, isolate and characterise the different epithelial cell compartments within the adult mammary gland. However, in all of these studies only a handful of markers were used to define and trace cell populations. Therefore, there is a need for an unbiased and comprehensive description of mammary epithelial cells within the gland at different developmental stages. To this end we used single cell RNA sequencing (scRNAseq) to determine the gene expression profile of individual mammary epithelial cells across four adult developmental stages; nulliparous, mid gestation, lactation and post weaning (full natural involution).
Project description:Non-lymphoid tissues (NLTs) harbour a pool of adaptive immune cells distinct from their counterparts in lymphoid tissues, and their development and phenotype remains largely unexplored. We used scRNA-seq to survey CD4+ T regulatory (Treg) and memory T (Tmem) cells in spleen, lymph nodes, skin and colon in an unbiased way, in mouse. This cross-tissues comparison allows us to obtain marker genes for immune populations in specific locations, as well as examine each population's heterogeneity. Additionally, a continuous phenotype of Treg migration can be modelled from the mouse data, unravelling the transcriptional stages through which these cells transition between tissues.
Project description:Chimeric embryos were generated to investigate the effect of Tal1 knockout in mouse embryos by single-cell RNA-sequencing. Tal1 is an essential transcription factor for the formation of the embryonic blood. Embryo chimerism permits the analysis of the effects of Tal1 knockout without the confounding effects of the absence of embryonic blood, which results in global developmental failures. Embryos were generated by blastocyst injection of tdTomato-labelled, Tal1-/- mouse embryonic stem cells into wild type embryos. After blastocyst harvest, cells were flow-sorted before 10X Genomics library preparation and single-cell RNA-sequencing.