{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Rufus Daw"],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16654"],"description":["This dataset consists of single-cell RNA sequencing (scRNA-seq) data derived from Peripheral Blood Mononuclear Cells (PBMCs) of two ischaemic stroke patients (P66 and P79). The study follows a longitudinal design to identify temporal changes in the systemic immune response, with samples collected at two timepoints: the acute in-patient stage (V1) and at 6-9 months follow-up (V3).  Here, four samples (P66_V1, P66_V3, P79_V1, P79_V3) were multiplexed using the 10x Genomics CellPlex protocol and processed as a single pool. Approximately 33,000 cells were loaded onto a 10x Chromium Controller using the Chromium Next GEM Single Cell 5' Reagent Kit. This specific library represents the Gene Expression (GEX) portion of a multi-modal immune profiling experiment.  The primary objective of this study was to assess the dynamics of the circulating immune compartment following ischaemic stroke."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sample Collection - Venous blood was drawn from ischaemic stroke patients within 96 h of stroke symptom onset or time last seen well, as well as at the 6-9-month follow-up timepoint. Blood was collected in ethylenediaminetetraacetic acid (EDTA) tubes and processed within 4 h.   To obtain peripheral blood mononuclear cells (PBMCs), EDTA blood was diluted 1:1 with phosphate-buffered saline (PBS) supplemented with 2% foetal bovine serum (FBS) and centrifuged at 1200 g for 15 min at 20°C in SepMate™ PBMC Isolation Tubes (StemCell Technologies, Vancouver, Canada) with Ficoll-Paque™ PLUS density gradient media (Cat. No. 17144003; Cytiva Life Sciences, Amersham, UK). Following centrifugation, the top supernatant layer containing PBMCs was transferred to 50 mL falcon tubes, diluted with 20 mL PBS + 2% FBS and centrifuged at 500 g for 10 min at 20°C. Supernatant was discarded, and the cell pellet was resuspended in ACK Lysing Buffer (Cat. No. A1049201; Gibco, Waltham, USA) for 5 min at room temperature. PBS (10 mL) + 2% FBS was added and tubes centrifuged at 500 g for 5 min at 20°C. The cell pellet was resuspended in 20 mL FBS + 2% FBS and cells were counted before centrifugation at 500 g for 10 min at 20°C. Cells were resuspended at a final density of 5-6 × 106 cells / 500 µL CryoStor® CS10 Freezing Medium (Cat. No. 100-1061; StemCell Technologies), and frozen at -80°C in CoolCell™ Cell Freezing Vial Containers (Cat. No. 15542771; Corning, New York, USA). Finally, PBMC aliquots were transferred to liquid nitrogen until ready for use.","Library Construction - Following reverse transcription, the emulsion was broken and barcoded cDNA was purified and amplified. The cDNA was then fragmented, end-repaired, and A-tailed. 5' Gene Expression libraries were constructed by adaptor ligation and sample index PCR using the Chromium Next GEM Single Cell 5' Reagent Kits (10x Genomics). The resulting libraries were size-selected and purified.","Sequencing - Sequencing Protocol: Sequencing was performed on an Illumina NovaSeq 6000 using an S1 flow cell, targeting ~35,000 reads per cell. The four biological samples (P66_V1, P66_V3, P79_V1, P79_V3) were multiplexed together in a single pooled library using the 10x Genomics CellPlex protocol.  Read Layout:  Read 1 (R1): 28 bp - Cell Barcode (16 bp) + UMI (12 bp) Index 1 (I1): 10 bp - Sample index Index 2 (I2): 10 bp - Sample index Read 2 (R2): 90 bp - cDNA insert (biological sequence)  Data Processing: Demultiplexing was performed using CellPlex oligo tags to assign individual cells to their respective biological samples (P66_V1, P66_V3, P79_V1, P79_V3).  Note on Raw Data Files: Because all four samples were sequenced together in a single pooled library, the raw FASTQ files (TW_GEX_S1_L001/L002_*.fastq.gz) are shared across all samples and contain reads from all four biological samples prior to demultiplexing. Sample-specific processed files (barcodes.tsv.gz, matrix.mtx.gz, features.tsv.gz) were generated after computational demultiplexing and contain only the cells assigned to each respective sample.","Nucleic Acid Extraction - Approximately 33,000 cells per pool (8250 per individual, per stage) were loaded onto the 10x Genomics Chromium Controller. Cell lysis and RNA release occurred immediately after encapsulation within Gel Beads-in-emulsion (GEMs). Reverse transcription was performed inside the GEMs to generate barcoded cDNA using the Chromium Next GEM Single Cell 5' Reagent Kits (10x Genomics), according to the manufacturer’s protocol."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Data pre-processing was performed following scanpy’s standard vignette (1e4 normalisation, log1p logarithmized data, filtered to top 2000 variable features). Manual expert annotation was provided by two independent immunologists at the Lydia Becker Institute of Immunology and Inflammation. Here, each immunologist was supplied with the top 200 differentially expressed genes per cluster and a UMAP of the data detailing the location of each cluster. Cells were annotated by consensus, per cluster. UMAPs overlayed with specific gene expression was provided to the immunologists on request."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Scanpy (hash  98de5d1a)","10xGenomics Chromium 3'v3","Illumina NovaSeq 6000","CryoStor® CS10 Freezing Medium (Cat. No. 100-1061; StemCell Technologies)","Chromium Next GEM Single Cell 5' Reagent Kits (10x Genomics)"],"study_type":["RNA-seq of coding RNA from single cells"],"species":["Homo sapiens"],"pubmed_authors":["Rufus Daw","Harry Deijnen"],"additional_accession":[]},"is_claimable":false,"name":"Longitudinal Single-Cell Transcriptomic Profiling of the Peripheral Immune Response in Ischaemic Stroke","description":"This dataset consists of single-cell RNA sequencing (scRNA-seq) data derived from Peripheral Blood Mononuclear Cells (PBMCs) of two ischaemic stroke patients (P66 and P79). The study follows a longitudinal design to identify temporal changes in the systemic immune response, with samples collected at two timepoints: the acute in-patient stage (V1) and at 6-9 months follow-up (V3).  Here, four samples (P66_V1, P66_V3, P79_V1, P79_V3) were multiplexed using the 10x Genomics CellPlex protocol and processed as a single pool. Approximately 33,000 cells were loaded onto a 10x Chromium Controller using the Chromium Next GEM Single Cell 5' Reagent Kit. This specific library represents the Gene Expression (GEX) portion of a multi-modal immune profiling experiment.  The primary objective of this study was to assess the dynamics of the circulating immune compartment following ischaemic stroke.","dates":{"release":"2026-03-05T00:00:00Z","modification":"2026-03-05T02:02:41.742Z","creation":"2026-02-13T13:32:23.345Z"},"accession":"E-MTAB-16654","cross_references":{"ENA":["ERP189110"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005684","EFO_0005518","EFO_0003816","EFO_0004184"]}}