Single cell RNA sequencing analysis of mouse skin samples with multiple conditions (10x scRNA-seq)
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ABSTRACT: Single cell RNA sequencing of embryonic mouse dorsolateral skin were microdissected. All live cells were collected and submitted in bulk for droplet-based cDNA library preparation (9000 cells/sample)
Project description:DNA barcodes can be used to identify single cells in a sequencing data space while optical codes can be used to track single live cells in an image data space. We have developed dual image and DNA (ID)-coding, which identifies individual single cells in both live image and sequencing data spaces. Samples provided here are relevant to proof-of-concept studies of ID-coding presented in the associated publication. DNA barcoded micro-particles were encapsulated in hydrogel droplets with or without single cells. The hydrogel droplets were then subjected to “single-droplet sequencing” where whole polyA-bearing nucleic acid components within a hydrogel droplet (i.e. mRNA from cells and synthetic DNA on beads) were concatenated by the same cell barcodes.
Project description:Purpose: Determine the layer-specific indentities and heterogeneity of innate lymphoid cells in the skin by single cell transcriptomic analysis Methods: Innate lymphoid cells were sorted from epidermis, dermis and subcutis of adult B6 mice and subjected to droplet based single-cell captureing and RNA isolation and library preparation. Results: 3,431, 3,356 and 1,061 innate lymphoid cells from epidermis, dermis, and subcutis were analyzed. Unsupervised clustering of the combined data set performed in Seurat identified 6 distinct ILC clusters. Conclusions: This study reveals skin layer-specific transcriptomic signature of innate lymphoid cells
Project description:Detailed description (other than provided below) of growth conditions, RNA preparation, cDNA synthesis and hybridization conditions can be found in the submitted paper.
Project description:Different lesion types were microdissected out from snap-frozen white matter and placed on visium slides. Optimization of RNA release and visualization with Nikon Ecclipse Ti2 were followed by barcoded recover, library preparation and sequencing.
Project description:This is a droplet-based single cell transcriptome data set from 13 human fetal livers (6-18 PCW) and 8 skin and kidney samples (6-12 PCW). It includes 199,642 cells with a mean detected gene number of 3000.
Project description:Comparison of Bacillus subtilis wild type and cshA mutant at exponential versus stationary phase. Detailed description (other than provided below) of growth conditions, RNA preparation, cDNA synthesis and hybridization conditions can be found in the submitted paper.
Project description:Droplet-based massively parallel single-cell RNA sequencing (scRNAseq) was performed by encapsulating sorted live CD45+ tumour-infiltrating cells into droplets and libraries were prepared using Chromium Single Cell 5′ Reagent Kits v2 according to the manufacturer’s protocol (10X Genomics). The generated scRNA-seq libraries were sequenced using an Illumina HiSeq2500.
Project description:Recently, combining single-cell RNA sequencing (scRNA-seq) with upstream cell preservation procedures such as cryopreservation or methanol fixation has become more common. By separating cell handling and preparation, from downstream library generation, scRNA-seq workflows are more flexible and manageable. However, the inherent transcriptomics changes associated with cell preservation and how they may bias further downstream analysis remain unknown. Here, we present a side-by-side droplet-based scRNA-seq analysis, comparing the gold standard – fresh cells – to three different cell preservation workflows: dimethyl sulfoxide based cryopreservation, methanol fixation and CellCover reagent. Cryopreservation proved to be the most robust protocol, maximizing both cell integrity and low background ambient RNA. Importantly, gene expression profiles from fresh cells correlated most with those of cryopreserved cells. Such similarities were consistently observed across the tested cell lines (R ≥ 0.97), monocyte-derived macrophages (R = 0.97) and immune cells (R = 0.99). In contrast, both methanol fixation and CellCover preservation showed an increased ambient RNA background and an overall lower gene expression correlation to fresh cells. Thus, our results demonstrate the superiority of cryopreservation over other cell preservation methods. We expect our comparative study to provide single cell omics researchers invaluable support when integrating cell preservation into their scRNA-seq studies.
Project description:Genome-wide comparative transcriptome analysis of the Bacillus subtilis parent strain PY79 and its yvfI derivative with yvfI::T10::spc (TEK1) grown in PA medium Detailed description of sample preparation and microarray conditions can be also found in Irigul et al to be submitted
Project description:To identify cAMP-dependent lncRNAs, we treated DIV16 primary hippocampal neurons with 50uM Forskolin or equal volume DMSO for 30 minutes prior to RNA isolation using Trizol and Total RNA sequencing. We submitted samples to Scripps Florida Genomics Core for library preparation and sequencing.