{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Carlos Torroja"],"organism":["Sus scrofa"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-13898"],"description":["The aim of the single cell RNASeq experiment is to explore the molecular changes in the progressing and regressing plaques of an atherosclerosis pig mode and specifically the long-term changes glycolysis pathway. To produce these glycolysis signatures, we randomized male and female pigs into two experimental groups: an actively progressing atherosclerosis group subjected to 15 months of HFHC diet (HFHC group f, n=4; m, n=2), and an LF/MTPi group fed 12 months of HFHC diet and then transitioned to low-fat diet with MTP inhibitor for three months (f, n=4; m, n=1). At the time of euthanasia, plaque from the distal 0.5 cm of the abdominal aorta was quickly processed and analyzed by scRNA-sequencing."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Library Construction - After reverse transcription and cDNA amplification, sequencing libraries were prepared using the Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics).","Nucleic Acid Extraction - Up to 16500  cells were loaded into each port of a Chromium Next GEM Chip G (10x Genomics).","Sample Treatment - BMS-212122 is a chemical inhibitor for Microsomal Triglyceride Transfer Protein (MTP) with the IUAPC name 9- {4- [2,5-Dimethyl- 4- ({[4'-(trifluoromethyl)-2-biphenylyl] carbonyl} amino)-1H-benzimidazol-1-yl] butyl}-N-(2,2,2-trifluoroethyl)-9H-fluorene-9-carboxamide.Atherosclerosis was induced in PCSK9D374Y transgenic minipigs (males) by feeding a standard pig feed supplemented with 20% w/w lard, 1.5% w/w cholesterol (HFHC), 0.7% NaCl w/w, from 3 until 15 months of age. During the first 6 months, the diet was also supplemented with 0.7% cholic acid. Animals were then either continued on the HFHC diet for 3 months (HFHC group) or changed to the standard pig feed with supplemented BMS-212122 (LF/MTPi group). The BMS-212122 dose was 1 mg/(kg·day)  during the first week and 0.5 mg/(kg·day) for the rest of intervention period.","Sequencing - ). Libraries were sequenced in paired-end reads using a HiSeq 4000 system (Illumina) and processed with RTA v1.18.66.3.","Sample Collection - Animals were euthanized and the abdominal aorta and iliac arteries were quickly isolated and opened. The atherosclerotic plaque was dissected from the distal 0.5 cm and proximal 0.5 cm of the right iliac artery. The media, adventitia, and perivascular tissue were manually removed and approximately 200-250 mg of plaque was selected, washed in HBSS, and minced. It was incubated for 60 minutes at 37 °C in 1 ml of an enzyme solution containing 1,5 mg/ml (8 U/ml) of elastase (LS002279, Worthington Biochemical), 2 mg/ml of liberase (05401119001, Sigma Aldrich) and 300 µg/ml of DNaseI (Sigma Aldrich, DN25). Non-digested tissue was disaggregated using a smooth-tipped Pasteur pipette. Reactions were stopped by adding PBS containing 0.5% bovine serum albumin (BSA). The suspension was passed through a 70 µm pore filter to remove aggregates. Cells were collected by centrifugation and  resuspended in 500 µl PBS+0.5% BSA. DAPI (final concentration, 1 µg/ml final ) and Draq5 (Thermo Scientific 65-0880-96; final concentration, 5 µM) were added to enable detection of viable and dead cells. Viable cells (Draq5 positive and DAPI negative) were sorted with a FACSAria Cell Sorter."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Sequence Alignment - FASTQ files for each sample were processed using 10X CellRanger software (v6.0.0), using Sus scrofa genome reference Ss11.1 (Ensembl gene build v109). Pseudogenes and small RNAs have been removed from the reference.","Data Transformation - Filtration and clusterization of cell barcodes were performed using Seurat and scater R packages running in R version 4.1.2. After visual assessment of violin plots with quality metrics, thresholds for high-quality barcode retention were as follows: number of UMIs from 1000 to 30000, number of genes detected from 600, percent of mitochondrial reads up to 15%, percent of ribosomal genes up to 30%, percent of hemoglobin reads up to 0.1%, cell UMIs / Total > 0.2%, less than 50% of reads are from top 50 expressed gene. For rigorous doublet identification, 2 approaches were used: DoubletFinder version 2.0.4 and scDblFinder version 1.12.0 with an assumed doublet rate of 15%. Both tools should confirm that a cell is a singlet to be kept. Gene filtration included the removal of mitochondrial and hemoglobin genes,  Dataset of aorta plaque was composed of 11 individual runs (6 pigs from the HFD group and 5 pigs from the MTP group) and yielded 26807 after bad quality cells and doublets removal (2560.5 and 2683 median number of cells per pig in HFD and MTP group, respectively). The cleaned and merged count matrices were log-normalized and scaled. Highly variable features (2000) were selected with vst settings. For the principal component analysis, the first 30 components were used. After clusterization (resolution 0.2), 10 clusters were identified and annotated based on cell markers from the literature.  The clusters were merged based on belonging to 4 main cell types that can be identified in atherosclerotic vessels as follows: \\\"Macro/Mono/DC\\\", \\\"SMC-derived\\\", \\\"Lymphocytes\\\", and “Endothelial cells”. Differential gene expression analysis was performed using the MAST package for each cluster against other clusters (min-pct is 0.3, log2FC difference is 0.25) and between conditions  within each main cluster (log2FC threshold is set to 0 to compare all genes, min.pct is set to 0.05 so the test is done only for meaningfully expressed genes). After FDR p-value adjustment, genes were considered statistically differently expressed with p-value adjusted < 0.05."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina HiSeq 4000"],"study_type":["RNA-seq of coding RNA from single cells"],"species":["Sus scrofa"],"pubmed_authors":["Carlos Torroja","Jacob Bentzon","Paula Nogales","Diana Sharysh"],"additional_accession":[]},"is_claimable":false,"name":"Regulation of glycolytic activity in multiple cell types explains the ability of FDG-PET to measure disease activity in atherosclerosis","description":"The aim of the single cell RNASeq experiment is to explore the molecular changes in the progressing and regressing plaques of an atherosclerosis pig mode and specifically the long-term changes glycolysis pathway. To produce these glycolysis signatures, we randomized male and female pigs into two experimental groups: an actively progressing atherosclerosis group subjected to 15 months of HFHC diet (HFHC group f, n=4; m, n=2), and an LF/MTPi group fed 12 months of HFHC diet and then transitioned to low-fat diet with MTP inhibitor for three months (f, n=4; m, n=1). At the time of euthanasia, plaque from the distal 0.5 cm of the abdominal aorta was quickly processed and analyzed by scRNA-sequencing.","dates":{"release":"2025-07-09T00:00:00Z","modification":"2025-07-23T20:55:24.453Z","creation":"2024-03-11T13:51:24.633Z"},"accession":"E-MTAB-13898","cross_references":{"ENA":["ERP158494"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005684","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0003969","EFO_0004184"]}}