biostudies-arrayexpressPetra Catalina SchwalieHomo sapiensscanpy, bescacellrangerhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14760The study investigates the interaction between rAAV and the innate immune system. We conducted single cell RNA-sequencing (scRNA-seq) of PBMCs from six healthy human donors, comparing samples taken either before or after 1, 4 or 24 hrs of incubation with rAAV8. As a control, scRNA-seq was performed on untreated whole blood incubated under the same culture conditions to consider the potential impact of the ex vivo culture on the blood cells.biostudies-arrayexpressNucleic Acid Extraction - GEM generation and library preparation was performed on the Chromium TM Controller (10X Genomics, Aventura, FL) using Single Cell 3’ dual index reagent kit v3.1 (10X Genomics) according to the manufacturer's instructions.Library Construction - Briefly, after GEM generation, a post GEM-reverse transcriptase cleanup and cDNA amplification was performed. The quality control and quantification of cDNA were assessed using a high sensitivity Bioanalyzer Chip (Agilent Technologies, Santa Clara, CA) before sample indexing using the Dual Index Kit TT Set A (10X Genomics). The final libraries were assessed for quality and concentration using a Bioanalyzer HS Assay kit (Agilent Technologies) and Qubit dsDNA HS assay kit (ThermoFisher, Waltham, MA).Sample Treatment - After incubation for 4 hrs or 24 hrs at 37°C in a 5% CO2 incubator, supernatants were harvested. Cytokine levels (IFN-γ, IL-8, TNF-ɑ, IL-2 and IL-6) in the supernatants were measured with the Human 5-plex array (Quanterix). PBMCs isolated from whole blood samples using EasySep Direct human PBMC isolation kit (Stemcell) were assessed for cell number and viability.Sequencing - A 2.5 nM equimolar pool of 10X 3’ scRNA-seq libraries were prepared prior to paired-end sequencing on a Novaseq 6000 (Illumina, San Diego, CA) using a 100 cycle paired end kit and S2 flow cell (Illumina). A read depth of 50 million reads per sample was targeted. The 10X 3’ single cell library constructs use standard Illumina P5 and P7 adaptors. The 16 base pair (bp) 10x barcodes and 12bp unique molecular index (UMI) are both sequenced in Read 1. The eight base pair sample index sequences incorporated as the sample index read 90 bp of the insert are sequenced in Read 2. TruSeq Read 1 and Read 2 are standard Illumina sequencing primer sites used in paired-end sequencing. Sequencing the libraries produces a standard Illumina BCL data output folder. Demultiplexing and fastq files are created using 421 the cellranger 6.0.2 mkfastq+count workflow v1.1 in Arvados.Sample Collection - Fresh whole blood from healthy human donors was collected in heparin lithium-coated blood collection tubes. Blood was plated in a 6-well plate and rAAV8 was added at a final concentration of 5e11 viral genome (vg)/ml. After incubation for 4 hrs or 24 hrs at 37°C in a 5% CO2 incubator, supernatants were harvested. PBMCs isolated from whole blood samples using EasySep Direct human PBMC isolation kit (Stemcell) were assessed for cell number and viability. For each sample, suspensions of 800 cells/μl were prepared as input for GEM (Gel beads in Emulsion) generation with a target recovery of 8,000 cells.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Demultiplexing and fastq files were created using the cellranger 6.0.2 mkfastq+count workflow v1.1 in Arvados. Fastq files were aligned to the human transcriptome (GRCh38) using CellRanger count v6.0.2 with the parameters ‘-- expect-cells = 6000’.Data Transformation - All cells showing >200 counts were further merged across all samples and processed with scanpy and the besca standard workflow. Filtering was performed with the parameters min_genes = 600, min_cells = 20, min_counts = 1000, n_genes = 6000, percent_mito = 0.15, max_counts = 40000. In brief, RNA counts were normalized per 10,000, the top most highly variable genes were selected, total gene and mitochondrial reads were regressed out, PCA was performed and the first 50 principal components were used for nearest-neighbor calculations and Leiden clustering, as well as for UMAP-based visualization. For annotating the samples, a first version of the analysis was run using bbknn for integration (with time as batch). Annotation was performed using besca’s sig-annot module and annotations transferred to a second version of the analysis with no integration/batch correction, which was used for all downstream analyses.UnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000RNA-seq of coding RNA from single cellsHomo sapiensPetra Catalina SchwaliefalseScRNA-seq of healthy human PBMC samples treated with recombinant AAV8 or controls, assessed at multiple time-pointsThe study investigates the interaction between rAAV and the innate immune system. We conducted single cell RNA-sequencing (scRNA-seq) of PBMCs from six healthy human donors, comparing samples taken either before or after 1, 4 or 24 hrs of incubation with rAAV8. As a control, scRNA-seq was performed on untreated whole blood incubated under the same culture conditions to consider the potential impact of the ex vivo culture on the blood cells.2025-07-05T00:00:00Z2025-01-20T22:10:44.006Z2025-01-20T22:10:44.006ZE-MTAB-14760EFO_0002944EFO_0004170EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0004184EFO_0003969