Project description:To delineate the potential molecular mechanisms underlying the communication between neutrophils and NK cells, we performed scRNAseq of the neutropenic bone marrow (12 months after transplantation of Cebpacre/+ Sbds +/+ or F/F cells). To this end, bone marrow cells were subsorted into HSC+MPP (LKS CD48-), HPC1 (LKS CD48+CD150-), B and T cells (B220+, CD3+), NK cells (NK1.1+ NKp46+) and a myeloid ‘rest’ fraction (B220-,CD3-,NKp46- and NK1.1-) and sorted fractions pooled together to obtain robust representation of all bone marrow cell types in the scRNAseq data
Project description:In this work, we compared different protocols to prepare single-cell suspensions used for scRNAseq and suggest an optimized dissociation protocol for mouse retina, which preserves cell morphology to a higher level leading to an overall increase of gene number per cell. We compared scRNAseq libraries generated with our optimized protocol to publicly available scRNAseq data of mouse retina. We further demonstrate a pipeline to reduce noise in scRNAseq caused by multiplets and ambient RNA.
Project description:In this work, we compared different protocols to prepare single-cell suspensions used for scRNAseq and suggest an optimized dissociation protocol for mouse retina, which preserves cell morphology to a higher level leading to an overall increase of gene number per cell. We compared scRNAseq libraries generated with our optimized protocol to publicly available scRNAseq data of mouse retina. We further demonstrate a pipeline to reduce noise in scRNAseq caused by multiplets and ambient RNA.
Project description:To comprehensively capture changes in retinal transcriptome for the LCA7 organoids compared to control, we performed single cell RNA-sequencing (scRNAseq) using the 10X Genomics platform. Retinal organoids at D150 of differentiation were dissociated for scRNAseq analysis. scRNAseq data revealed significant dysregulation of specific photoreceptor genes between control and LCA7 organoids, as well as mutation-specific differences in various genes, including CRX, RCVRN, ARR3, and AIPL1.
Project description:scRNAseq of HPMCs isolated ex vivo from 3 Patients. Targeted scRNASeq was applied using BD onco Panel with a additional genes. Purpose of the experiment was to characterize HPMC phenotype ex vivo and compare with the phenotype observed in RNAseq of HPMCs treated with IL17A and TNF.
Project description:Single cell transcriptomic analyses (scRNAseq) of hepatocytes and liver endothelial cells (L-EC) have revolutionized the understanding of the spatial architecture of liver structure and function. The spatial alignment of L-EC and hepatocytes is pivotal for liver function in health and disease given that L-EC act as instructive gatekeeper of nearby hepatocytes including the maintenance of liver metabolic zonation in a Wnt-dependent manner. Advancing liver biology beyond the ’transcript-centric’ view of scRNAseq analyses is presently restricted by the limited resolution of proteomics and genome-wide techniques to analyse post-translational modifications. Here, by combining spatial cell sorting methodology with transcriptomic and quantitative proteomic/phospho-proteomic analyses, we established the first functionally and spatially-resolved proteome landscape of the liver endothelium, yielding deep mechanistic insight into zonated vascular signalling mechanisms. Phosphorylation of receptor tyrosine kinases (RTK) was detected preferentially in the central vein area resulting in an atypical enrichment of tyrosine phosphorylation. Prototypic biological validation of the identified strong phosphorylation gradient of the vascular RTK Tie1 by blockade resulted in the rapid peri-central dysregulation of the L-EC transcriptome. Notably, the expression of Wnt9b in L-EC was discovered as Tie receptor controlled with reciprocal regulation by FoxO1 and STAT3 transcription factors. Genetic inactivation of Tie1 in L-EC or antibody blockade resulted in reduced liver regeneration following partial hepatectomy with reduced Wnt ligand and Wnt target gene expression, including Axin2, Sox9, Tbx3 and Lgr5. Taken together, the study has yielded unparalleled insight into the spatial organization of L EC signalling and discovered a vascular Tie-Wnt signalling axis as regulator of liver function. The employed spatial sorting technique followed by phospho-proteomic analysis may be employed as a universally adaptable strategy for the spatial phosphoproteomic analysis of scRNAseq data-defined relevant cellular (sub)-populations.
Project description:Single cell transcriptomic analyses (scRNAseq) of hepatocytes and liver endothelial cells (L-EC) have revolutionized the understanding of the spatial architecture of liver structure and function. The spatial alignment of L-EC and hepatocytes is pivotal for liver function in health and disease given that L-EC act as instructive gatekeeper of nearby hepatocytes including the maintenance of liver metabolic zonation in a Wnt-dependent manner. Advancing liver biology beyond the ’transcript-centric’ view of scRNAseq analyses is presently restricted by the limited resolution of proteomics and genome-wide techniques to analyse post-translational modifications. Here, by combining spatial cell sorting methodology with transcriptomic and quantitative proteomic/phospho-proteomic analyses, we established the first functionally and spatially-resolved proteome landscape of the liver endothelium, yielding deep mechanistic insight into zonated vascular signalling mechanisms. Phosphorylation of receptor tyrosine kinases (RTK) was detected preferentially in the central vein area resulting in an atypical enrichment of tyrosine phosphorylation. Prototypic biological validation of the identified strong phosphorylation gradient of the vascular RTK Tie1 by blockade resulted in the rapid peri-central dysregulation of the L-EC transcriptome. Notably, the expression of Wnt9b in L-EC was discovered as Tie receptor controlled with reciprocal regulation by FoxO1 and STAT3 transcription factors. Genetic inactivation of Tie1 in L-EC or antibody blockade resulted in reduced liver regeneration following partial hepatectomy with reduced Wnt ligand and Wnt target gene expression, including Axin2, Sox9, Tbx3 and Lgr5. Taken together, the study has yielded unparalleled insight into the spatial organization of L EC signalling and discovered a vascular Tie-Wnt signalling axis as regulator of liver function. The employed spatial sorting technique followed by phospho-proteomic analysis may be employed as a universally adaptable strategy for the spatial phosphoproteomic analysis of scRNAseq data-defined relevant cellular (sub)-populations.