Single-cell and bulk RNA-seq of sorted and micro-dissected mouse bone marrow and fetal liver cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE89378 | GEO | 2017/11/01
SECONDARY ACCESSION(S): PRJNA352039
REPOSITORIES: GEO
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Project description:We here systematically studied the interaction network of bone marrow cells. To this end, we micro-dissected many small interacting structures (cell doublets, triplets etc.) into single cells, and sequenced their mRNAs, to infer cell identity. After grouping the cells into cell types (based on the single-cell transcriptomes), we identified actual physical interactions that occurred more, or less, than what would be expected by chance. We compared the micro-dissected data to sorted hematopoietic stem cells.
2016-06-24 | GSE76983 | GEO
Project description:We here systematically studied the interaction network of bone marrow cells. To this end, we micro-dissected many small interacting structures (cell doublets, triplets etc.) into single cells, and sequenced their mRNAs, to infer cell identity. After grouping the cells into cell types (based on the single-cell transcriptomes), we identified actual physical interactions that occurred more, or less, than what would be expected by chance. We compared the micro-dissected data to sorted hematopoietic stem cells. After mild dissociation of the bone marrow, we micro-dissected many small interacting structures (cell doublets, triplets etc.) into single cells, and sequenced their mRNAs. In addition, single hematopoietic stem cells were sorted (Lineage- Kit+ Sca1+ CD150+ CD48-) to sequence their transcriptome. In detail, the bone marrow was mildly flushed. Structures composed of about 10 to 20 cells were set apart. With needles and a micro-dissection microscope, we trimmed of smaller structures from these big structure. These smaller structures are mainly two to four cells attached together. These small structures were then further micro-dissected to single cells. The goal of doing these sequential dissections was to keep track of which cells were interacting with which. Each micro-dissected cell sample contains RNA-seq data for 96 single cells. Each single cells received a different barcode that allow us to entangle them, and to produce the processed .coutt.csv file. Each sorted hematopoietic stem cell sample represents a single cell. The processed data file expdata_BMJhscC.csv contains transcript counts for all analyzed cells.
2016-06-24 | E-GEOD-76983 | biostudies-arrayexpress
Project description:We here systematically studied the interaction network of small intestine crypt cells.We first created a reference of cell types based on single-cell mRNA sequencing, which we used to create virtual doublets and triplets. These virtual combinations were then used to train a machine learning algorithm (random forests) in order to infer the cell types present in the actual picked doublets or triplets. We then counted the number of interactions in between defferent cell types, and compared it to label permution simulation to identify interactions that happens more or less than expected by chance.
2018-04-16 | GSE103516 | GEO
Project description:We have applied cryo-tomography and proteomics analyses to centrosomes isolated from young lamb thymus– a convenient source of quiescent and differentiated cells. Those analyses have allow us to better depict the protein organization joining the two centrioles in such a cellular system, in the form of a disperse network of fibers and an amorphous density, both parts linked together. Protein composition of the centrosome have been compared with published data on centrosome extracted from cell culture , and presence of various protein reported implicated in the disengagement and dissociation processes questioned. C-NAP1, cohesin smc1 and furthermore condensin smc4 and ncapd2 have been localized on the amorphous density, however DNA hasn't. Subtomogram averaging processing have been applied to the centriolar wall, both on the proximal microtubule triplets and distal doublets; results have been compared to the already published structures of centriolar wall triplet from distant species basal bodies.
2019-11-08 | PXD003928 | Pride
Project description:BACKGROUND:Systematic cancer screening has led to the increased detection of pre-malignant lesions (PMLs). The absence of reliable prognostic markers has led mostly to over treatment resulting in potentially unnecessary stress, or insufficient treatment and avoidable progression. Importantly, most mutational profiling studies have relied on PML synchronous to invasive cancer, or performed in patients without outcome information, hence limiting their utility for biomarker discovery. The limitations in comprehensive mutational profiling of PMLs are in large part due to the significant technical and methodological challenges: most PML specimens are small, fixed in formalin and paraffin embedded (FFPE) and lack matching normal DNA. METHODS:Using test DNA from a highly degraded FFPE specimen, multiple targeted sequencing approaches were evaluated, varying DNA input amount (3-200 ng), library preparation strategy (BE: Blunt-End, SS: Single-Strand, AT: A-Tailing) and target size (whole exome vs. cancer gene panel). Variants in high-input DNA from FFPE and mirrored frozen specimens were used for PML-specific variant calling training and testing, respectively. The resulting approach was applied to profile and compare multiple regions micro-dissected (mean area 5 mm2) from 3 breast ductal carcinoma in situ (DCIS). RESULTS:Using low-input FFPE DNA, BE and SS libraries resulted in 4.9 and 3.7 increase over AT libraries in the fraction of whole exome covered at 20x (BE:87%, SS:63%, AT:17%). Compared to high-confidence somatic mutations from frozen specimens, PML-specific variant filtering increased recall (BE:85%, SS:80%, AT:75%) and precision (BE:93%, SS:91%, AT:84%) to levels expected from sampling variation. Copy number alterations were consistent across all tested approaches and only impacted by the design of the capture probe-set. Applied to DNA extracted from 9 micro-dissected regions (8 PML, 1 normal epithelium), the approach achieved comparable performance, illustrated the data adequacy to identify candidate driver events (GATA3 mutations, ERBB2 or FGFR1 gains, TP53 loss) and measure intra-lesion genetic heterogeneity. CONCLUSION:Alternate experimental and analytical strategies increased the accuracy of DNA sequencing from archived micro-dissected PML regions, supporting the deeper molecular characterization of early cancer lesions and achieving a critical milestone in the development of biology-informed prognostic markers and precision chemo-prevention strategies.
| S-EPMC7672910 | biostudies-literature
Project description:The aim of this BBSRC-funded project is to develop laser-capture microdissection (LCMD) to isolate small cell clusters in different regions of arabidopsis embryos at different stages of development; to develop RNA amplification procedures on dissected tissue sampes; and to use DNA microarray techniques to investigate global transcriptional differences between samples. Cryosectioned embryos of ecotype Col-O of globular, heart and torpedo stage were used to isolate cell clusters from the apical and basal regions, for RNA isolation and amplification. !Samples will be provided as T7-primed cDNA, with three biological replicates for each tissue to be analysed. Each replicate comprises cDNA from pooled tissue samples from ca. 15 embryos. The experimental details have been discussed with Sean May et al. at NASC. Experimenter name = Stuart Casson and Matthew Spencer Experimenter phone = 0191 374 7356 Experimenter fax = 0191 374 2417 Experimenter institute = Durham University Experimenter address = Integrative Cell Biology Laboratory Experimenter address = School of Biological Sciences Experimenter address = Durham University Experimenter address = South Road Experimenter address = Durham Experimenter zip/postal_code = DH1 3LE Experimenter country = UK Keywords: organism_part_comparison_design; development_or_differentiation_design
2007-01-08 | GSE5730 | GEO
Project description:Identify gene changes in articular cartilage of the medial tibial plateau (MTP) at 2, 4 and 8 weeks after destabilisation of the medial meniscus (DMM) in mice. Compare our data with previously published datasets to ascertain dysregulated pathways and genes in osteoarthritis (OA).RNA was extracted from the ipsilateral and contralateral MTP cartilage, amplified, labelled and hybridized on Illumina WGv2 microarrays. Results were confirmed by real-time polymerase chain reaction (PCR) for selected genes.Transcriptional analysis and network reconstruction revealed changes in extracellular matrix and cytoskeletal genes induced by DMM. TGF? signalling pathway and complement and coagulation cascade genes were regulated at 2 weeks. Fibronectin (Fn1) is a hub in a reconstructed network at 2 weeks. Regulated genes decrease over time. By 8 weeks fibromodulin (Fmod) and tenascin N (Tnn) are the only dysregulated genes present in the DMM operated knees. Comparison with human and rodent published gene sets identified genes overlapping between our array and eight other studies.Cartilage contributes a minute percentage to the RNA extracted from the whole joint (<0.2%), yet is sensitive to changes in gene expression post-DMM. The post-DMM transcriptional reprogramming wanes over time dissipating by 8 weeks. Common pathways between published gene sets include focal adhesion, regulation of actin cytoskeleton and TGF?. Common genes include Jagged 1 (Jag1), Tetraspanin 2 (Tspan2), neuroblastoma, suppression of tumourigenicity 1 (Nbl1) and N-myc downstream regulated gene 2 (Ndrg2). The concomitant genes and pathways we identify may warrant further investigation as biomarkers or modulators of OA.
| S-EPMC4373757 | biostudies-literature
Project description:Refrigerated storage of red cell concentrates before transfusion is associated with progressive alterations of red blood cells (RBC). Small RBC (type III echinocytes, sphero-echinocytes, and spherocytes) defined as storage-induced micro-erythrocytes (SME) appear during pretransfusion storage. SME accumulate with variable intensity from donor to donor, are cleared rapidly after transfusion, and their proportion correlates with transfusion recovery. They can be rapidly and objectively quantified using imaging flow cytometry (IFC). Quantifying SME using flow cytometry would further facilitate a physiologically relevant quality control of red cell concentrates. RBC stored in blood bank conditions were stained with a carboxyfluorescein succinimidyl ester (CFSE) dye and incubated at 37°C. CFSE intensity was assessed by flow cytometry and RBC morphology evaluated by IFC. We observed the accumulation of a CFSE high RBC subpopulation by flow cytometry that accounted for 3.3 and 47.2% at day 3 and 42 of storage, respectively. IFC brightfield images showed that this CFSE high subpopulation mostly contains SME while the CFSE low subpopulation mostly contains type I and II echinocytes and discocytes. Similar numbers of SME were quantified by IFC (based on projected surface area) and by flow cytometry (based on CFSE intensity). IFC and scanning electron microscopy showed that ≥95% pure subpopulations of CFSE high and CFSE low RBC were obtained by flow cytometry-based sorting. SME can now be quantified using a common fluorescent dye and a standard flow cytometer. The staining protocol enables specific sorting of SME, a useful tool to further characterize this RBC subpopulation targeted for premature clearance after transfusion.
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Project description:The aim of this BBSRC-funded project is to develop laser-capture microdissection (LCMD) to isolate small cell clusters in different regions of arabidopsis embryos at different stages of development; to develop RNA amplification procedures on dissected tissue sampes; and to use DNA microarray techniques to investigate global transcriptional differences between samples. Cryosectioned embryos of ecotype Col-O of globular, heart and torpedo stage were used to isolate cell clusters from the apical and basal regions, for RNA isolation and amplification. !Samples will be provided as T7-primed cDNA, with three biological replicates for each tissue to be analysed. Each replicate comprises cDNA from pooled tissue samples from ca. 15 embryos. The experimental details have been discussed with Sean May et al. at NASC. Experimenter name = Stuart Casson and Matthew Spencer; Experimenter phone = 0191 374 7356; Experimenter fax = 0191 374 2417; Experimenter institute = Durham University; Experimenter address = Integrative Cell Biology Laboratory; Experimenter address = School of Biological Sciences; Experimenter address = Durham University; Experimenter address = South Road; Experimenter address = Durham; Experimenter zip/postal_code = DH1 3LE; Experimenter country = UK Experiment Overall Design: 27 samples were used in this experiment
2008-06-14 | E-GEOD-5730 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the SubSeries listed below.
2016-11-01 | GSE89379 | GEO