Project description:Purpose: Analysis at the single cell level of the CD4+ and CD8+ Lymphocytes T extracted and PBMCs from healthy human donor Methods: Single Cell RNAseq performed using the 10X genomics platform
Project description:Single-cell RNA-sequencing (scRNA-Seq) is widely used to characterize immune cell populations. However, mRNA levels correlate poorly with expression of surface proteins, which are well established to define immune cell types. CITE-Seq (cellular indexing of transcriptomes and epitopes by sequencing) utilizes oligonucleotide-tagged antibodies to simultaneously analyze surface phenotypes and transcriptomes. Considering the high costs of adding surface phenotyping to scRNA-Seq, we aimed to determine which of 188 tested CITE-Seq antibodies can detect their antigens on human peripheral blood mononuclear cells (PBMCs), a commonly interrogated cell population in immunology, and find the optimal concentration for staining. The recommended concentration was optimal for 76 antibodies, whereas staining quality of 7 antibodies improved when the concentration was doubled. 33 and 8 antibodies still worked well when the concentration was reduced to 1/5 or 1/25, respectively. 64 antigens were not detected at any antibody concentration. Optimizing the antibody panel by removing antibodies not able to detect their target antigens and adjusting concentrations of the remaining antibodies could enable a cost reduction of almost 50%. In conclusion, our data are a resource for building an informative and cost-effective panel of CITE-Seq antibodies and use them at their optimal concentrations in future CITE-seq experiments on human PBMCs.
Project description:We performed scRNAseq of PBMCs from three idiopathic multicentric Castleman Disease (iMCD) patients with paired flare and remission samples
Project description:We used scRNAseq to profile CD71/CD24low fetal liver erythroid progenitor cells isolated by 2 distinct methods: FACS and immunomagnetic isolation. Cells from both isolation methods were hashtagged using Biolegend mouse hashtag antibodies and library prepped together on the 10X chromium platform with the 3'RNA v3 kit. We also performed CITE-seq to profile proteogenomic expression of CD117 and CD71 on lineage-depleted mouse fetal liver erythroid progenitor cells. CITE-seq was performed through a separate library prep on the 10X chromium platform with the 3'RNAv3 kit.
Project description:Monocyte heterogeneity is poorly understood in tumors. Here we utilize scRNAseq to profile monocyte and myeloid cell diversity in tumor and non-tumor-bearing mice.
Project description:Midbrain organoid derived from human induced pluriopotent stem cells a new model of Parkinson's Disease. Organ specific organoids can be used to model many diseases, however little is know about these models. We created a flow cytometry workflow to identify cell types in midbrain organoids. We then selected four populations and sorted these populations: Neurons1(CD24++), Neurons2(CD56++), Astrocytes and Radial Glia, using FACS and the antibody intensity gates defined by our workflow. We then performed scRNAseq on the sorted population and identified cell types within the sorted populations.
Project description:We report here the analysis of young and aged hematopoietic stem and progenitor cells (HSPCs) by transcriptional profiling (bulk RNA-Seq, CITE-Seq) and chromatin profiling (ATAC-Seq, scATAC-Seq). We use here a Clca3a1 as a surface marker to further fractionate HSPCs. HSPCs were sorted by flow cytometry. During the staining procedure for flow cytometry, the antibody cocktail also included antibodies labelled with oligos (antibody-derived tags =ADTs) which can later be analyzed by the 10x Chromium controller.