ScRNA-seq of barcode labelled cells of the postnatal mouse cochlea
Ontology highlight
ABSTRACT: E7.5 embryos were injected with LV-EF1A-H2B-tdTomato-30N barcode using ultrasound guided injections. Pups were collected at postnatal stage and single cell RNA sequenced using 10X sequencing.
INSTRUMENT(S): Nova Seq S6000, Illumina NovaSeq 6000
Project description:LV-EF1A-H2B-tdTomato-30N barcode virus was injected into the amniotic cavity (AC) of embryos at E7.5, using ultrasound guided in-utero nano-injections. E9.5 and E10.5 whole embryos were collected, dissociated, and sorted for tdTomato. Sorted cells were subjected to single cell RNA sequenced using 10X Genomics sequencing.
Project description:We have captured 100,000 single cells for single-cell RNAseq from whole mouse embryos during gastrulation and organogenesis, spanning days 6.5 to 8.5 of development, including embryonic and extraembryonic tissues. Cells were sampled every six hours, providing a continuous molecular characterisation of these processes. Cell libraries were prepared using the 10X Genomics Chromium platform.
Project description:Single-cell RNA-seq analysis of mouse CD4 T cells to understand the function of HDAC1 for pathogenic Th2 cells. Wildtype and HDAC1 KO cell from several sorted populations are covered. Hashtag oligos were used to enable multiplexing before analysis on a 10x chromium.
Project description:We obtained human embryonic and fetal lungs from 5-22 pcw for scRNAseq and scATACseq analysis. To focus on epithelial differentiation and region specialization, we deeply sampled 15, 18, 20 and 22 pcw lungs and separated proximal and distal regions while leaving lungs at 5, 6, 9 and 11 pcw intact. These cell samples (except for one at 6pcw) were split and processed for both scRNAseq and scATACseq.
Project description:Aberrant activation of the canonical Wnt pathway underlines the development and growth of intestinal tumors. The Apc-min (multiple intestinal neoplasia) mouse strain carries a single nucleotide mutation in one allele of the Apc tumor suppressor gene. Random deactivation of the second allele occurs during the life and results in the formation of multiple adenomas in the small intestine with occasional colonic occurence. The Defa6 promoter is active in adult small intestinal Paneth cells and its activation has been observed also in human colonic adenomas. To investigate the nature of Defa6+ colon adenoma cells, we used the Apc-min Rosa26-tdTomato Defa6-iCre mouse strain. In this strain, cells with an active Defa6 promoter are marked by expression of a red fluorescent protein (tdTomato). We performed gene expression profiling of Defa6-tdTomato+ cells isolated from mouse colon tumors.
Project description:Intracellular calcium levels are finely tuned through intricate actions of a number of channels and transporters, including those at key endocellular stores, e.g. endoplasmic reticulum (ER), lysosomes, and mitochondria. Along with the highly homologous genes Inositol 1,4,5-trisphosphate (IP3) receptor type 1 (ITPR1) and 2 (ITPR2), ITPR3 encodes the IP3 receptor (IP3R), a key player in intracellular calcium release in animals. Here we report the first cases of ITPR3 defects in man leading to a primarily dysimmune phenotype. In four unrelated patients of diverse ethnicity, and suffering from a complex immunodeficiency syndrome, we report the same de novo pathogenic variant - c.7570C>T; p.Arg2524Cys - in ITPR3. Clinically, recurrent severe infectious episodes of viral and bacterial origins, features of ectodermal dysplasia and that of Charcot-Marie-Tooth disease were paramount. The identified variant does not affect gene transcription, yet it was structurally predicted and biologically proven to disrupt proper protein folding and function in vivo. This eventually leads to defective Calcium flux in patient cells, dysregulation of mitochondrial function and a broad dysimmune phenotype characterized primarily by a profound CD4 T cell lymphopenia associated with quasi absence of naïve CD4 and CD8 cells, itself mirrored by an increase in cognate memory cells. The Calcium signaling defect was recapitulated ex vivo through the introduction of this single variant in Jurkat cells. Moreover, site-directed mutagenesis displayed the exquisite sensitivity of Arg2524 to any amino acid change. In conclusion, a single unique recurrent de novo variant in ITPR3 leads to a novel syndromic immunodeficiency.
Project description:We performed a detailed analysis of the in vitro differentiation of induced pluripotent stem cells to macrophages and dendritic (chromatin accessibility data).
Project description:Comprehensive molecular cancer studies have extensively characterized most primary and some metastatic tumor types over the last decade. In prostate cancer, the most common tumor type in men, genomic studies have been most notably conducted for primary and metastatic tumors that had progressed under androgen deprivation therapies (ADT) to castration-resistant disease (CRPC). More recent studies have also looked at genetic alterations in a smaller number of prostate cancers frequently associated with neuroendocrine trans-differentiation. For this tumor type, it’s becoming clear that the complexity of genomic alterations does not allow an accurate assessment of the transformed phenotype. To overcome these hurdles, we show in patient-derived xenograft models how disease progression emerges at single-cell resolution and how pharmacologic perturbation can revert this process. Given this approach, we were able to investigate disease progression and androgen independence at single cell level: in our xenograft models, we show how tumor cell subpopulation progress along the trajectory from androgen-sensitive to insensitive disease.
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).