Single cell RNA sequencing of stem cell-derived retinal ganglion cells
ABSTRACT: 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: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 performed 3' single-cell RNA-seq using the 10X Genomics Chromium (version 1 chemistry) system on ~19,000 undifferentiated human IPSCs to explore the cellular heterogeneity of a seemingly homogeneous cell population.
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: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:To explore the interactions between the range of maternal and fetal placental cell types present, we profiled the transcriptomes of more than 50,000 single cells from matched first trimester samples of maternal blood and decidua, as well as fetal cells from the placenta itself. RNA-seq was done using the standard 10x chromium v2 chemistry.
Project description:Raw sequencing data used in the paper "A biological-computational cell lineage discovery platform based on duplex MIPs" (doi: https://doi.org/10.1101/191296) involving single cells from the YUCLAT melanoma patient and DU145 cell line which cultured in standart condition as recommend. The single cells were prepared by CellCellector for DU145 and FACS for YUCLAT, amplified by RepliG WGA kit . Targeting sequencing library is cronstructed with duplex Molecular Inversion Probes wtih the protocol as described in the paper. Illumina NextSeq is used to sequence these libraries.
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 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 transcriptome profiling using a 3' droplet-based platform (Chromium,10x Genomics) of CD11b+ cells isolated from the spleen of control and tumor-bearing mice, treated or not with IFN gene therapy.