biostudies-arrayexpressDavid JohnMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15635Transcriptomic (snRNA-seq) analyses were performed on epigenetically repressed ZBTB16 in cardiac aging in mice.biostudies-arrayexpressSequencing - Library quantification and quality assessment were performed using Bioanalyzer Agilent 2100 using a High Sensitivity DNA chip (Agilent Genomics). Indexed libraries were equimolarly pooled and sequenced on Illumina NovaSeq 6000 using paired-end 26x98 bp as sequencing mode by GenomeScan (Leiden, Netherlands).Sample Collection - To isolate nuclei for transcriptomic analysis, hearts were perfused with 1X DPBS in vivo, snap-frozen in liquid nitrogen and stored at -80°C until usage. Eight mice hearts were sequenced (n=4 per group, two females and two males, respectively).Nucleic Acid Extraction - For the nuclei isolation, one female and one male heart per group were pooled and processed together. The hearts were minced in pre-filtered homogenization buffer containing 250 mM sucrose (1623637, Sigma-Aldrich), 25 mM KCl (AM9640G, Invitrogen), 5 mM MgCl2 (AM9530G, Invitrogen), 10 mM Tris buffer pH 8.0 (AM9855G, Invitrogen), 1 mM DTT (P2325, Thermo-Fisher Scientific), 1X protease inhibitor (11697498001, Roche), 0.6 U/µL Ambion RNase inhibitor (AM2682, Thermo-Fisher Scientific), 0.1% Triton (T8787, Sigma-Aldrich) and 2% BSA (A8022, Sigma-Aldrich) in ultra-pure DNase/RNase-free distilled water (10977035, Thermo-Fisher Scientific). Cells were disrupted with seven strokes of a loose pestle in a glass dounce homogenizer. The homogenized solution was filtered using a pre-wetted 40 µm cell strainer followed by a pre-wetted 20 µm cell strainer. After centrifugation (500 g at 4°C for 8 minutes), the cell pellet was re-suspended in sorting buffer containing 2% BSA, 0.6 U/µL Ambion RNase inhibitor, 1 mM DTT in DPBS. 7AAD (420403, Biolegend) positive nuclei were separated from cell debris using the FACS-Aria III instrument (BD Biosciences; Nozzle size 100 microns) into collection buffer containing 2% BSA, 1 U/µL Ambion RNase inhibitor, 1 mM DTT in DPBS. Sorted nuclei were washed with 0.04% BSA in DPBS.Library Construction - Nuclei suspensions were loaded on a 10X Chromium Controller (10X Genomics) according to manufacturer’s protocol based on the 10X Genomics proprietary technology. All snRNA-seq libraries were prepared using Chromium Single Cell 3′ v3 Reagent Kit (10X Genomics) according to manufacturer’s protocol. Briefly, the initial step consisted in performing an emulsion where individual nuclei were isolated into droplets together with gel beads coated with unique primers bearing 10X cell bar-codes, UMI (unique molecular identifiers) and poly(dT) sequences. Reverse transcription reactions were engaged to generate barcoded full-length cDNA followed by the disruption of emulsions using the recovery agent and cDNA clean up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific). Total cDNA was amplified using a Biometra Thermocycler TProfessional Basic Gradient with 96-Well sample block (98 °C for 3 min; cycled 14×: 98 °C for 15 s, 67 °C for 20 s, and 72 °C for 1 min; 72 °C for 1 min; held at 4 °C). Amplified cDNA product was cleaned with the SPRIselect Reagent Kit (Beckman Coulter). Indexed sequencing libraries were constructed using the reagents from the Chromium Single Cell 3′ v3 Reagent Kit, as follows: fragmentation, end repair and A-tailing; size selection with SPRIselect; adaptor ligation; post-ligation cleanup with SPRIselect; sample index PCR and cleanup with SPRI select beads.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Library preparation was performed as previously described11 and single-nucleus RNA sequencing results were processed with the 10X Genomics Cell Ranger pipeline (7.2.0). The command “count” was employed to aligned the raw reads to the mouse reference genome (GRCm38-2019). Following the best practices for single-nucleus RNA sequencing, the option “include-introns” was activated during the analysis. To mitigate ambient RNA contamination, the ‘remove-background’ module of CellBender (0.3.0) was applied to the raw data counts12.Data Transformation - The downstream processing and analysis were conducted with Scanpy (1.9.6)13 operated within Python 3.9.18. Our downstream analysis excluded genes expressed in less than 3 cells. We further removed cells with insufficient gene counts (<250 UMI) and with an excess of mitochondrial RNA counts (>10%). Doublet detection was performed using Scrublet (0.2.3) assuming an expected doublet rate of 6%. Additionally, the top 5% of cells with the highest number of UMI counts were discarded. After normalization of the data to total counts and a log-transformation, the most variable genes across the different batches were identified to performed dimensionality reduction and integration through principal component analysis and BBKNN batch correction approach (1.6.0) using 3 neighboring cells within batches. The uniform manifold approximation and projection (UMAP) embeddings were computed to visualize the data. Cell clustering was conducted using the Leiden algorithm with a resolution parameter of 0.3. Identified clusters were automatically annotated by using CellTypist (1.6.2., Human Heart model V1.0.)14. The annotation was further cross-checked with gene markers previously identified for the different cell types of the heart15–17. To identify differentially expressed genes between the two conditions, the Wilcoxon rank-sum test and the Benjamini-Hochberg correction method was applied using the “rank_genes_groups” function of Scanpy. To delve deeper into potential cell-to-cell interactions among the identified cell types in the different conditions, we employed CellChat (2.1.1)18 within R 4.3.3., by following the recommended “Comparison of multiple datasets” tutorial18. Single nuclei ATAC sequencing was performed as previously described19.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000single nucleus RNA sequencingMus musculusDavid JohnfalseEndothelial expression of ZBTB16 protects against cardiac agingTranscriptomic (snRNA-seq) analyses were performed on epigenetically repressed ZBTB16 in cardiac aging in mice.2025-10-24T00:00:00Z2026-05-27T13:36:38.603Z2025-09-25T20:43:39.12ZE-MTAB-15635ERP180637EFO_0002944EFO_0004170EFO_0004917EFO_0009809EFO_0005518EFO_0003816EFO_0004184