Project description:Fresh blood samples were collected from 8 PD patients and 6 age-matched healthy controls. Then, peripheral blood mononuclear cells (PBMCs) were isolated by Percoll density gradient centrifugation. Next, CD19+ B cells were sorted using fluorescence-activated cell sorting (FACS). Single-cell 5’ gene expression libraries and V(D)J enriched libraries were prepared according to the standard protocols provided by the 10x Genomics Chromium Single Cell Immune Profiling Solution. Finally, single-cell 5’ gene expression and V(D)J libraries were sequenced on Illumina Noveseq 6000, providing 150 bp paired-end reads.
Project description:Alzheimer’s disease (AD) is the most common dementia worldwide with no current treatment and poorly understood molecular mechanisms. RNA expression studies suggest that AD is determined by diverse molecular events. Single cell technology allows comprehensive investigation of gene expression and cell specific changes in high precision of large number of brain cells. Computational analysis can reveal affected specific cell type marker genes and generate classification of the samples and gene networks. Here, we extensively characterized altered gene expression changes in post mortem brain samples of 2 individuals with AD (the brain samples originating from 3 brain regions, BA41/42,BA 6/8 and anterior hippocampal cortex). Wed detected significant changes in astrocyte marker genes. The samples were taken from patients in Braak stage III-IV. We found significant gene expression changes between the AD compared to the 2 control sequenced samples. We found significant changes in oligodendrocyte, microglia and astrocyte marker genes and several specific genes that were altered including the neuronal differentiation factor SOX2. Overall, our study thus reinforces a growing body of evidence implicating glia in AD, and highlights the need for greater mechanistic understanding of neuron-glia interactions in AD.
Project description:The Merkel cell carcinoma cell line (MCC) WaGa was sequenced using the 10x Genomics 3' Chromium v2.0 platform in order to analyse transcriptional heterogeneity on a single cell level
Project description:Methods: Cells isolated from the whole brains of naive wild type, infected wild type, and infected CCR2-DTR mice were sort-purified on live CD45hi cells from five biological replicates and pooled. Sort-purified cells were then processed using the 10X Genomics Chromium Controller. Cell suspensions were loaded onto the Chromium Single Cell A Chip for cell lysis and barcoding. RNA from individual cells was reverse transcribed and sequencing libraries prepared using the Chromium Single Cell 3’ Library Kit v2 following the manufacturers protocol. Samples were sequenced using an Illumina NextSeq 550 with standard 10X Genomics Configuration (26 bp x 98 bp). After sequencing, raw bcl files were processed using the cellranger mkfastq command for sample demultiplexing and conversion to .fastq files, followed by cellranger count for cell barcode and UMI deconvolution as well as mapping to the respective reference genome. Processed digital gene expression matrices were imported into R studio for analysis using the Seurat package. Samples were aligned along common sources of variation and compared using canonical correlation analysis to identify unique clusters of cells within the samples. Marker genes for each sample and cluster were identified and used for generation of downstream plots within the Seurat package. All packages are maintained to be best in class and are regularly updated to their most recent release. Results: 10 distinct cell clusters were identified and we found that two populations were found at increased proportions post-infection but were substantially reduced in the monocyte-depleted animals (CCR2-DTR). We confirmed that these populations to be monocytes and monocyte-derived cells, and valicated their immunoregulatory molecules by RT-qPCR analysis.
Project description:Over 16,000 nuclei were isolated from human postmartum brain frozen prefrontal cortex samples of alcoholic and control individuals. Libraries were prepared with 10X Genomics platform and sequenced using NovaSeq 6000.
Project description:The Merkel cell carcinoma cell line (MCC) WaGa was sequenced without and under HDAC inhibitor treatment using the 10x Genomics 3' Chromium v2.0 platform. The treatment induced changes were analysed on a single cell level.
Project description:To compare the performance of Illumina and BGI sequencing technologies for high-throughput single cell sequencing, four Chromium single cell libraries of the following human cell types: Induced Pluripotent Stem Cells (hIPSC), cultured Trabecular MeshWork Cells (TMWC) and Peripheral Blood Mononuclear Cells (PBMCs), were sequenced on Illumina sequencers (NextSeq 500, NovaSeq 6000) and a BGI sequencer (MGISEQ-2000). The technologies were benchmarked based on sequencing quality, characterisation of cell populations within samples and for specific single cell analyses such as variant calling and detection of guide RNAs from pooled CRISPR screens.
Project description:Differential expression analysis of human brain samples with Alzheimer disease versus healthy control samples using an Agilent custom expression microarray.