Project description:To analyse the transcriptome landscape of differentiating cardiomyocytes, GFP labeled cardiac cells were purified by fluorescent activated cell sorting using the TinC*>GFP transgenic line, and expression profile analysed by deep sequencing.
Project description:Background Proteomic characterization of microglia has been limited by low yield and contamination by non-microglial proteins by magnetic-activated cell sorting (MACS) enrichment strategies. To determine whether a fluorescent-activated cell sorting (FACS)-based strategy overcomes these limitations, we compared microglial proteomes of MACS and FACS-based purified CD11b+ microglia in order to identify core sets of microglial proteins in adult mouse brain tissue. Results Quantitative multiplex proteomics by tandem mass tag mass spectrometry (TMT-MS) of MACS-enriched (N = 5) and FACS-purified (N = 5) adult wild-type CD11b+ microglia identified 1,791 proteins, of which 953 were differentially expressed indicating significant differences between both approaches. While the FACS-purified microglia proteome was enriched with cytosolic, endoplasmic reticulum and ribosomal proteins involved in protein metabolism and immune system functions, the MACS-enriched microglia proteome was enriched with proteins related to mitochondrial function and synaptic transmission. As compared to MACS, the FACS microglial proteome showed strong enrichment for canonical microglial proteins while neuron, astrocyte, and oligodendrocyte proteins were decreased. Interestingly, we observed enrichment of endothelial specific proteins in the FACS microglia proteome. By comparing FACS-purified microglia proteomes with transcriptomes, we observed highly concordant as well as highly discordant proteins that were abundant at the protein level but low at the transcript level. Conclusions We demonstrate that TMT-MS proteomics of FACS-purified adult microglia is superior to column-based enrichment approaches, resulting in purer and highly-enriched microglial proteomes. We also define core sets of highly-abundant adult microglial proteins including Moesin (Msn) that can guide future studies.
Project description:3 subtypes of cortical projection neurons were purified by fluorescence-activated cell sorting at 4 different stages of development from mouse cortex. A detailed description of the data set is described in Arlotta, P et al (2005). Keywords = corticospinal motor neuron callosal corticotectal cortex development FACS
Project description:Differences of metabolism-related gene expression profiles in human ESCs and ESC-derived purified cardiomyocytes were analyzed and successfully identified. Human ESCs and ESC-derived purified cardiomyocytes were used for this experiment.
Project description:We sequenced mRNA from HFSCs that were FACS purified (CD34+alpha-6 Integrin+ cells) from p21 mouse epidermis, total epidermal cells from p21 mice and HFSCs cultured for 14 days in 3C cultures, 3 biological replicates each. We sequenced mRNA from different cell populations that were FACS-purified from HFSC cultures (also known as 3C cultures), 3 biological replicates each. We performed two independent comparisons: 1. Comparison of FACS-purified cells grown in standard 3C cultures: HFSCs (CD34+ alpha-6 Integrin+) and non-HFSCs (CD34neg alpha-6 Integrin+). 2. Comparison of total cells in 3C cultures treated for 48 hours with dorsomorphin (BMP inhibitor that causes depletion of CD34+ alpha-6 integrin+ cells in 3C cultures) or with cyclopamine Shh inhibitor that causes enrichment of CD34+ alpha-6 integrin+ cells in 3C cultures).
Project description:Differences of metabolism-related gene expression profiles in human ESCs and ESC-derived purified cardiomyocytes were analyzed and successfully identified.
Project description:This SuperSeries is composed of the following subset Series:; GSE2039: FACS purified cortical projection neurons; GSE17783: Analysis of gene expression in FACS-purified cortical projection neurons using Affymetrix 430 2.0 microarrays Experiment Overall Design: Refer to individual Series
Project description:The goal of this experiment was to evaluate the expression profile of cardiomyocytes freshly isolated from zebrafish embryos. Here we report mRNA abundance measurements for zebrafish cardiomyocytes at 20 hours post fertilization (hpf). We found that cardiomyocytes isolated from zebrafish embyros are enriched for orthologues of genes abundant in mammalian iPS-derived cardiomyocytes and other in vitro models of cardiomyocytes. It was also enriched with genes required for cardiac development in zebrafish and mammalian models. Using a rapid-throughput CRISPR-Cas9 based G0 screening pipeline, we tested 50 abundant transcription factors derived from this profile, rediscoved transcription factors known to be required for cardiac development, and found a novel role for zbtb16a in cardiac development.
Project description:The identification of novel cardiomyocyte-intrinsic factors that support ventricular function will expand the number of candidate genes and therapeutic options for heart failure, a leading cause of death worldwide. Here, we demonstrate that a conserved RNA-binding protein RBPMS2 is required for ventricular function in zebrafish and for myofibril organization and the regulation of intracellular calcium dynamics in zebrafish and human cardiomyocytes. A differential expression screen uncovered co-expression of rbpms2a and rbpms2b in zebrafish cardiomyocytes. Double knockout embryos suffer from compromised ventricular filling during the relaxation phase of the cardiac cycle, which significantly reduces cardiac output. Evaluating rbpms2-null embryos with splicing-sensitive differential expression analysis, quantitative PCR, and in situ hybridization revealed differential alternative splicing of cardiomyopathy genes including myosin binding protein C3 (mybpc3) and phospholamban (pln). Cardiomyocytes in double mutant ventricles and those derived from RBPMS2-null human induced pluripotent stem cells exhibit myofibril disarray and calcium handling abnormalities. Taken together, our data suggest that RBPMS2 performs a conserved role in regulating alternative splicing in cardiomyocytes, which is required for sarcomere organization, optimal calcium handling, and cardiac function.