biostudies-arrayexpressCarlos TorrojaMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15242Aging is the main risk factor for atherosclerotic cardiovascular disease, a condition caused by immune cells that leads to the buildup of plaques in arteries and is the top cause of death worldwide. Earlier studies on premature aging diseases have linked this condition to problems with A-type lamins, important parts of the cell’s nuclear envelope. In this study, we looked at how aging changes the levels of lamin A/C in white blood cells and how changing these levels in blood-forming cells affects their behavior and the development of atherosclerosis Atheroprone Ldlr-/- mice were used as recipients in BM transplantation (BMT) experiments. During 4 weeks after BMT, mice were on standard diet for BM reconstitution and then placed on a high-fat diet (HFD) for 6 weeks to induce hypercholesterolemia and atherosclerosis development. Mouse studies were conducted with male and female 8-14 week old, except for BMT with lamin A/C-null BM, which was obtained from 3–4 week old. Experimental groups are: 1) WT BM, mice transplanted with WT BM donors 2) Lmna-/- BM, mice transplanted with BM from donors lacking lamin A/C ubiquitously. 3) Lmnatg BM, mice transplanted with BM from Lmnatg silent donors. 3) Lmna-OE BM, mice transplanted with BM from Lmnatg Overexpressing donors.biostudies-arrayexpressNucleic Acid Extraction - Cells were recounted and their viability checked on a Countess III cell counter (Thermofisher). Cells were loaded into each port of a Chromium Next GEM Chip G (10x Genomics) for single cell isolation and cDNA reverse transcription and amplificationSample Collection - Aortas from Ldlr-/- mice reconstituted with Lmna-/-, Lmna+/+, Lmna-OE, or Lmnatg BM were thoroughly perfused with PBS to remove blood. The aortic arch and thoracic aorta were dissected, cleaned of perivascular fat and opened longitudinally. Aortas from 3-5 transplant recipients of the same genotype were pooled, with both male and female donors included in each pool to minimize potential sex-related bias. Aortas were digested at 37°C for 15 minutes in DMEM supplemented with collagenase A (6.25 mg/ml, Roche), dispase II (6.25 mg/ml, Roche), DNase I (62.5 μg/ml; Roche), and elastase (1.717 U/ml, Sigma). Cells were treated with 0.25% trypsin-EDTA (Gibco) for 5 minutes at 37°C, pelleted and resuspended in sorting buffer. Purified cells were sorted to filter cells according to size and complexity (FSC, SSC), and then select viable nucleated cells (Hoechst 33342+, TO-PRO-3−) for sorting, discarding dead cells and debris.Sequencing - Libraries were sequenced in paired-end reads using a HiSeq 4000 system (Illumina) and processed with RTA v1.18.66.3Library Construction - Sequencing libraries were created using the Next GEM Single cell 3’Library preparation kit v3.1 (10x Genomics) and indexed using the Chromium i7 Multiplex kit (10x Genomics) following the manufacturer instructionsOrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Single cell identification and transcriptome profile was obtained using the 10X CellRanger pipeline (v6.1.1) and the mouse mm10 genome reference (ensembl gene build v98, mm10-2020-A). Quantification matrices were processed using scater and seurat R packages. In brief, QC stats were obtained for Mitocondria, Ribosome, Hemoglobine, IGs and TRs using scater. Cells were filtered with the following conditions: 1000 > total_counts > 35000 & detected_genes > 500 & MT_content < 25 & top_50 > 65, HBB_percent < 0.1 & Cell_Fraction > 0.2. Clustering was perform using Seurat. All protocol with its detailed parametrization can be found here: https://github.com/LAB-VA-CNIC/sc-lmna.TG.OE.HFD.UnknownTranscriptomicsGenomicsProteomicsIllumina HiSeq 2500RNA-seq of coding RNA from single cellsMus musculusMarta AmorósCarlos TorrojaVicente AndrésfalseLamin A/C expression in hematopoietic cells declines during human aging and constrains atherosclerosis in miceAging is the main risk factor for atherosclerotic cardiovascular disease, a condition caused by immune cells that leads to the buildup of plaques in arteries and is the top cause of death worldwide. Earlier studies on premature aging diseases have linked this condition to problems with A-type lamins, important parts of the cell’s nuclear envelope. In this study, we looked at how aging changes the levels of lamin A/C in white blood cells and how changing these levels in blood-forming cells affects their behavior and the development of atherosclerosis Atheroprone Ldlr-/- mice were used as recipients in BM transplantation (BMT) experiments. During 4 weeks after BMT, mice were on standard diet for BM reconstitution and then placed on a high-fat diet (HFD) for 6 weeks to induce hypercholesterolemia and atherosclerosis development. Mouse studies were conducted with male and female 8-14 week old, except for BMT with lamin A/C-null BM, which was obtained from 3–4 week old. Experimental groups are: 1) WT BM, mice transplanted with WT BM donors 2) Lmna-/- BM, mice transplanted with BM from donors lacking lamin A/C ubiquitously. 3) Lmnatg BM, mice transplanted with BM from Lmnatg silent donors. 3) Lmna-OE BM, mice transplanted with BM from Lmnatg Overexpressing donors.2025-11-06T00:00:00Z2025-11-06T18:09:35.512Z2025-06-18T15:41:08.824ZE-MTAB-15242ERP173618EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0003816EFO_0004184