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Isolated nuclei from frozen tissue are the superior source for single cell RNA-seq compared with whole cells

ABSTRACT: Isolated nuclei from frozen tissue are the superior source for single cell RNA-seq compared with whole cells

PROVIDER: PRJNA938031 | ENA |

REPOSITORIES: ENA

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Isolated nuclei from frozen tissue are the superior source for single cell RNA-seq compared with whole cells

Project description:The isolation of intact single cells from frozen tissue is a challenge due to the mechanical and physical stress inflicted upon the cell during the freeze-thaw process. Ruptured cells release ambient RNA into the cell suspension, which can become encapsulated into droplets during droplet based single cell RNA-seq library preparation methods. The presence of ambient RNA in droplets has been suggested to impact data quality, however there have been limited reports on single cell RNA-seq data from frozen tissue. Here, we compare the results of single cell RNA-seq derived from disaggregated cells from frozen brain tissue with single nuclei RNA-seq derived from purified nuclei of identical tissue using the 10X Genomics Chromium 3’ gene expression assay. Our results indicated that presence of ambient RNA in the cell suspension resulted in single cell RNA-seq data with a 25-fold lower gene count, a 5-fold lower UMI count per cell and a 2-fold lower fraction of reads per cell compared with single nuclei RNA-seq data. Cell clustering with the single cell RNA-seq data was unable to resolve the heterogeneity of brain cell types. Our conclusion is that nuclei from frozen tissue are the superior substrate for single cell transcriptome analysis.

2023-02-28 | GSE225915 | GEO

Large-scale secretome analyses unveil a superior immunosuppressive phenotype from umbilical cord stromal cells compared to other adult mesenchymal stromal cells

Project description:Mesenchymal stromal cells (MSCs) with regenerative and immunomodulatory potential are being investigated as a potential therapeutic tool for cartilage lesions. MSCs express a wide variety of bioactive molecules including cytokines, trophic factors, and proteases, which act in a paracrine fashion to modulate the tissue microenvironment. Yet, little is known about the divergence of these signalling molecules between MSCs populations from adult or young tissues. This makes it challenging to decide the optimal source of MSCs for a specific clinical application. In this study, we investigated cell secretomes from cultured human stromal cells harvested from Hoffa’s fat pad (HFPSCs), synovial membrane (SMSCs), umbilical cord (UCSCs) and cartilage (ACs) by quantitative LC-MS/MS proteomics. We also performed multiplex protein arrays and functional assays to compare the constitutive immunomodulatory capabilities of different MSCs. Proteins involved in extracellular matrix degradation and inflammation such as MMPs, IL-17, and complement factors were significantly downregulated in UCSCs compared to other cell types. Additionally, we found enhanced expression of TGF-β1 and PGE2 in UCSCs supernatants. UCSCs were superior in inhibiting peripheral blood mononuclear cells proliferation, migration and TNF-α and IFN-γ secretion compared to ACs, HFPSCs and SMSCs. Although all cell types could repress HLA-DR surface expression and cytokine release by activated macrophages, only UCSCs significantly blocked IL-6 and IL-12 production. Our data demonstrate that stromal cells from umbilical cords display superior anti-inflammatory and immunosuppressive properties than stromal cells from adult tissues. This Allogeneic cell source could potentially be considered as an adjuvant therapy for articular cartilage repair.

2019-01-29 | PXD011762 | Pride

Nanoparticle Protein Coronas Differentially Interrogate Proteomes with Superior Performance Compared to Conventional Deep Proteome Workflows

Project description:The superior performance of a multiple NP workflow was shown compared to high-complexity, state-of-the-art, high-pH fractionation strategies in terms of depth and precision. We also evaluate the link between the physicochemical properties of NPs and their differential protein selectivity.

2022-02-24 | PXD028634 | Pride

Nanoparticle Protein Coronas Differentially Interrogate Proteomes with Superior Performance Compared to Conventional Deep Proteome Workflows

2022-02-24 | PXD022285 | Pride

FIN-Seq: Transcriptional profiling of specific cell types in frozen archived tissue from the human central nervous system

Project description:FIN-Seq is a method that allows transcriptional profiling of specific cell types in frozen postmortem human CNS tissues. Briefly, nuclei from frozen CNS samples are isolated, fixed, and immunolabeled with antibodies for prospective isolation. The RNA from the FACS isolated nuclei are extracted and an RNA sequencing library is generated for subsequent sequencing.

2019-10-10 | GSE130143 | GEO

Single Nucleus DNA Sequencing of Flow Sorted Archived Frozen and Formalin Fixed Paraffin Embedded Solid Tumors

Project description:Archived tissues are a vast resource of annotated clinical samples. To expand their use for translational studies, we optimized flow sorting and sequencing of single nuclei from archived frozen and FFPE tumor tissues. Our methods, which include isolation and preparation of intact nuclei suitable for library preparations, quality control (QC) metrics for each step, and a single cell sequencing bioinformatic processing and analysis pipeline, were validated with flow sorted nuclei from matching frozen and FFPE ovarian cancer surgical samples and a sequencing panel of 553 amplicons targeting single nucleotide and copy number variants in genes of interest.

2024-06-20 | GSE270364 | GEO

snRNA-seq of nuclei isolated with microvessel-enrichment procedure applied to frozen post-mortem brain tissue

Project description:snRNA-seq of nuclei isolated with microvessel-enrichment procedure applied to frozen post-mortem brain tissue

| PRJNA940797 | ENA

Single nuclei RNA-sequencing of adult human hearts

Project description:We report single nuclei RNA-sequencing data from two frozen left ventricular biopsies from a patient with dilated cardiomyopathy, providing a perspective on the cell types of the adult human failing heart.

2020-11-11 | GSE161153 | GEO

Aspergillus niger exposed to combinations of carbon source, nitrogen source, nitrogen concentration, and culture pH

Project description:Microarray analysis of Aspergillus niger under conditions with differing combinations of carbon source, nitrogen source, nitrogen concentration, and culture pH Fermentor cultures were grown in minimal medium (MM) at a constant temperature of 30 ± 0.5 ºC and with differing combinations of carbon source (either 277.5 mM glucose or 333.0 mM xylose), nitrogen source (NH4Cl or NaNO3) and nitrogen concentration (4x: 282.4 mM; 8x: 564.8 mM), and pH (pH4 or pH5) of the medium (M. Braaksma, A.K. Smilde, M.J. van der Werf, P.J. Punt, submitted for publication). At different time points samples were collected, quenched immediately in methanol at -45 ºC and centrifuged at -20 ºC to remove supernatant. Part of the biomass was frozen into liquid nitrogen and stored at -80 ºC for microarray analysis. For each of the 16 culture conditions one sample was selected for microarray analysis; samples were collected either around the time point carbon source depleted or a considerable time (~24 h) after carbon souce depletion. In addition some technical duplicates were included.

2010-04-08 | GSE17329 | GEO

Multi-modal single-cell and whole-genome sequencing of minute, frozen specimens to propel clinical applications [slideseq]

Project description:We provide 5' and/or 3' single cell and single nuclei RNA sequencing data for matched comparisons of profiling from fresh (single cells) and frozen (single nuclei) samples of a non small cell lung cancer sample, a cutaneous melanoma sample , and a primary uveal melanoma sample. We also provide matched samples processed with published dissociation protocols (*TST and *CST) for two samples as comparison. Next, we profiled three sequential biopsies (one pre-treatment and two on-treatment) from the KEYNOTE-001 trial by 5' single-nuclei RNA sequencing from frozen specimens. For these three datapoints we provide matched SlideSeq V2 spatial transcriptomic data. Finally, we provide 5' single nuclei RNA-sequencing data for 7 liver-metastatic uveal melanoma samples profiled at pre, on, and post treatment timepoints with MEK-inhibitor selumetinib. For all samples profiled with 5' capture we also provide TCR receptor information. This study provides a framework for the application of single nuclei RNA-sequencing on clinical grade specimens, and demonstrates the feasibility of this approach to dissect the biology of archival specimens or rare diseases.

2022-10-19 | GSE216055 | GEO

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