C62c6565-9a12-4cd2-bd1f-1256b3a905ac - samples
Ontology highlight
ABSTRACT:
PROVIDER: EGAD00001006110 | EGA |
REPOSITORIES: EGA
Similar Datasets
Project description:Pancreas and spleen were microdissected from wild type (C57B6/SV129 background) E14.5 embryos and P0 newborn pups. Pools of 30-60 tissues were used. Tissues were digested for 1 hour at 37°C in HBSS/0.1% collagenase A/20 µg/ml DNase I, followed by dissociation of the cell clusters into single cells using a non-enzymatic dissociation medium (Sigma). Single cell suspensions were blocked with mouse IgGs and anti-CD16/32 Abs and immunostained with biotin-anti-CD11b (Biolegend) and RPE-conjugated goat anti-CCR2 (R&D Systems), followed by Cy5.5 conjugated streptavidin. CD11b+CCR2+ cell were then isolated by FACS sorting. mRNA from purified cells was prepared using the RNAeasy kit (Qiagen) and run on a MouseWG-6 v2.0 Expression BeadChip.
2017-12-06 | E-MTAB-5427 | biostudies-arrayexpress
Project description:We report single cell gene expression profiles for Ewing Sarcoma cell lines CHLA9, CHLA10, and TC71. Cell lines were harvested and prepared as single cell suspensions with high viability. Library prep was performed with the 10X Genomics v3.1 3' kit. All samples were sequenced on the illumina HiSeq 3000 with a 28+8+100 configuration. CHLA9 and CHLA10 libraries were re-sequenced on the Illumina NovaSeq with a 28+8+91 configuration. Counts were processed with cellranger v3.1.0. Both the raw data and the cellranger output are provded.
2020-04-11 | GSE146221 | GEO
Project description:In this study, we performed single-cell transcriptional profiling of human embryonic and fetal gut samples obtained from 9 human embryos spanning ages 6-10 PCW and three regions (duo-jejunum, ileum and colon). Additionally, we profile mucosal biopsies from the terminal ileum of healthy children aged 4-12 years (n = 8) as well as a group of children newly diagnosed with Crohn’s disease (CD) (n = 7), a common form of IBD. Tissue samples were treated using an enzymatic dissociation protocol and single cell suspensions were then processed using the 3’v2 10x Genomics Chromium workflow. In a subset of samples, the intestinal epithelial cell fraction was enriched by performing magnetic bead sorting for EPCAM. In total, we generate single cell transcriptomes of ~90,000 primary human intestinal cells providing a rich resource and detailed roadmap. Using this data as well as scRNAseq profiles of human fetal gut derived organoids we describe embryonic and fetal epithelium composition, trace their differentiation dynamics and signaling partners, and provide links to regenerating Crohn’s disease epithelium.
2020-12-22 | E-MTAB-8901 | biostudies-arrayexpress
Project description:Tissue dissociation, mechanical and/or enzymatic, is a method of disaggregating 3D connective tissue to release cells for downstream single cell analysis. Without proper controls for tissue dissociation experiments, potential artifacts can be mistaken for baseline tissue data. We hypothesized that enzymatic (collagenase/hyaluronidase) compared to mechanical dissociation leads to dissociated related artifacts not found in endogenous tumor tissue. To distinguish artifacts between enzymatic and mechanical dissociation, we compared separate enzymatic and mechanical dissociated samples with baseline samples from healthy and tumor patient whole blood, melanoma and head and neck squamous cell carcinoma (HNSCC) tumor tissues in flow cytometry, RNA sequencing and CODEX experiments. In bulk sorted tumor tissue, there is a significant difference between enzymatic and mechanical digested samples in cell stress, heat shock proteins and cell death related genes. These genes were also significantly upregulated in enzymatic over mechanical samples in the single cell RNA sequencing HNSCC tissue data. Fibroblasts and CD39+CD103+ CD8 T cells from tumor tissue, appear to require enzymatic digestion for efficient release from tissue compared to the mechanical digested samples. CD39+CD103+ CD8 tumor infiltrating lymphocytes (TILs) show different phenotypes depending on dissociation technique and tumor tissue. In tumor tissue, CD3+CD8+CD69+ T cells and gene expression were elevated in the enzymatic samples over the mechanical and baseline frozen tissue. Tissue dissociation techniques and tissue analysis from downstream applications have the potential for dissociation related artifacts that can mislead both protein and RNA sequencing data analysis in cancer research.
2023-09-19 | GSE243360 | GEO
Project description:Tissue dissociation, mechanical and/or enzymatic, is a method of disaggregating 3D connective tissue to release cells for downstream single cell analysis. Without proper controls for tissue dissociation experiments, potential artifacts can be mistaken for baseline tissue data. We hypothesized that enzymatic (collagenase/hyaluronidase) compared to mechanical dissociation leads to dissociated related artifacts not found in endogenous tumor tissue. To distinguish artifacts between enzymatic and mechanical dissociation, we compared separate enzymatic and mechanical dissociated samples with baseline samples from healthy and tumor patient whole blood, melanoma and head and neck squamous cell carcinoma (HNSCC) tumor tissues in flow cytometry, RNA sequencing and CODEX experiments. In bulk sorted tumor tissue, there is a significant difference between enzymatic and mechanical digested samples in cell stress, heat shock proteins and cell death related genes. These genes were also significantly upregulated in enzymatic over mechanical samples in the single cell RNA sequencing HNSCC tissue data. Fibroblasts and CD39+CD103+ CD8 T cells from tumor tissue, appear to require enzymatic digestion for efficient release from tissue compared to the mechanical digested samples. CD39+CD103+ CD8 tumor infiltrating lymphocytes (TILs) show different phenotypes depending on dissociation technique and tumor tissue. In tumor tissue, CD3+CD8+CD69+ T cells and gene expression were elevated in the enzymatic samples over the mechanical and baseline frozen tissue. Tissue dissociation techniques and tissue analysis from downstream applications have the potential for dissociation related artifacts that can mislead both protein and RNA sequencing data analysis in cancer research.
2023-09-19 | GSE243359 | GEO
Project description:The interspersed anatomic distribution of endothelial cells (ECs) and their dependence on microenvironmental cues have been major challenges in studying EC function and response to (patho)physiological stimuli in vivo. Combining EC-specific translating ribosome affinity purification (EC-TRAP) with RNA sequencing provides an accurate in vivo snapshot of tissue-specific EC expression profiles, and maintains a high degree of sensitivity to detect low abundant transcripts while limiting changes in gene expression profiles due to enzymatic tissue dissociation required to generate single cell suspensions for FACS sorting or single cell analysis. In vitro HUVEC data was obtained to ensure short-term cycloheximide treatment, as used for in vivo experiments, did not alter global gene expression.
2019-10-10 | GSE138628 | GEO
Project description:The interspersed anatomic distribution of endothelial cells (ECs) and their dependence on microenvironmental cues have been major challenges in studying EC function and response to (patho)physiological stimuli in vivo. Combining EC-specific translating ribosome affinity purification (EC-TRAP) with RNA sequencing provides an accurate in vivo snapshot of tissue-specific EC expression profiles, and maintains a high degree of sensitivity to detect low abundant transcripts while limiting changes in gene expression profiles due to enzymatic tissue dissociation required to generate single cell suspensions for FACS sorting or single cell analysis. In addition to demonstrating EC heterogeneity under physiologic conditions, the in vivo host response to lipopolysaccharide (LPS) revealed the induction of expression programs associated with a native defense response, with marked differences across vascular beds.
2019-10-10 | GSE138629 | GEO
Project description:Animal R.401 was infused with ex-vivo expanded T-regulatory cells. After adoptive transfer, PBMC were obtained and stained with surface antibodies. Single cell libraries from sorted populations were generated using a UMI-based droplet-partitioning platform (10X Genomics) and sequenced using a NextSeq 500 (Illumina). Resulting reads were processed using Cellranger software (10X Genomics) and downstream analysis was performed using Monocle6,7 using a negative binomial model of distribution with fixed variance.
2019-10-24 | GSE139200 | GEO
Project description:Massively parallel single-cell RNA sequencing can precisely resolve cellular diversity in a high-throughput manner at low cost, but unbiased isolation of intact single cells from complex tissues, such as adult mammalian brains, is challenging. Here, we integrate sucrose-gradient assisted nuclei purification with droplet microfluidics to develop a highly scalable single-nucleus RNA-Seq approach (sNucDrop-Seq), which is free of enzymatic dissociation and nuclei sorting. By profiling ~18,000 nuclei isolated from cortical tissues of adult mice, we demonstrate that sNucDrop-Seq not only accurately reveals neuronal and non-neuronal subtype composition with high sensitivity, but also enables in-depth analysis of transient transcriptional states driven by neuronal activity, at single-cell resolution, in vivo.
2017-12-07 | GSE106678 | GEO
Project description:RNA-seq of embryonic mammary progenitor cell lines. Embryonic mammary progenitor cell (eMPC) clones were derived from E12.5-stage mammary organs. After microdissection, mammary primordial 3 was cultured on thick basement membrane extract for 4 weeks. After enzymatic dissociation eMPC swere plated and expanded in 2D culture to obtain a pool of eMPCs. eMPC cells were subject to single cell sorting using FACS. Clones were expanded as single-cell derived clones (eMPC1, 2, etc.) to create the eighteen single cell-derived clones described in this study.
2018-11-30 | E-MTAB-6856 | biostudies-arrayexpress