biostudies-arrayexpressNils Jonathan TrostGallus galluscellranger (6.0.2), cellranger-arc (2.0.2)scikit-learn (0.20.1), scrublet (0.2), Seurat (4.3.0.1), rliger (1.0.1)https://www.ebi.ac.uk/biostudies/studies/E-MTAB-17505This study used droplet-based snRNA-seq and joint snRNA-seq and snATAC-seq multiome to profile the gene expression of single nuclei in developing chicken ovaries across three timepoints, from embryonic day (E) 17 to E20. Fertilized chicken eggs (Gallus gallus) were purchased from Granja Santa Isabel and incubated at 37.5 °C in a humidified atmosphere until the required developmental stage. The day when eggs were incubated was considered E0. Single-cell barcoding and library preparation were performed using Chromium Single Cell 3' Reagent Kits (v3 chemistry) for snRNA-seq only and Chromium Single Cell Multiome ATAC + Gene Expression Reagent kits (v1) for multiome experiments with the Chromium Controller instrument (10x Genomics). Sequencing of the libraries was carried out on the Illumina Nbiostudies-arrayexpressLibrary Construction - Nuclei were processed using Chromium Single Cell ATAC Reagent kits (v1) and the Chromium Controller instrument (10x Genomics; RRID:SCR_019326), following the manufacturer's guidelines. The process included tagmentation, single cell barcoding, and library preparation. The libraries were then amplified through 10 PCR cycles and quantified using a Qubit Fluorometer (Thermo Fisher Scientific; RRID:SCR_018095). The average fragment size of the libraries was determined using a Fragment Analyzer (Agilent; RRID:SCR_019417).Sample Collection - Frozen tissue was homogenized on ice in a buffer containing sucrose, KCl, MgCl2, Tris-HCl (pH 8), IGEPAL, DTT, Murine RNase Inhibitor, SUPERase-In, and cOmplete Protease Inhibitor Cocktail. The tissue was disrupted by trituration and/or using a micropestle. After a brief incubation, unlysed tissue debris was removed by low-speed centrifugation (100g for 1 minute at 4°C). The supernatant was then centrifuged at 400g for 4 minutes to separate the nuclei (pellet) from the supernatant. Nuclei were washed once or twice in the homogenization buffer and resuspended in a storage buffer containing sucrose, KCl, MgCl2, Tris-HCl (pH 8), Murine RNase Inhibitor, SUPERase-In, and cOmplete Protease Inhibitor Cocktail. If needed, the nuclei were filtered using 40 µm Flowmi strainers. NSequencing - Sequencing was performed on a NextSeq 500/550 (Illumina; RRID:SCR_016381) with 34 cycles for both Read 1 and Read 2, 8 cycles for the i7 index, and 16 cycles for the i5 index.Nucleic Acid Extraction - RNA was extracted from the cytoplasm extracts or nuclei suspensions by mixing them with RLT buffer (supplemented with 40 mM DTT) and 100% ethanol in a 2:7:5 ratio. The RNA was then purified using the RNeasy Micro Kit from Qiagen. The quality of the extracted RNA was assessed using a Fragment Analyzer (Advanced Analytical), and all samples had RNA quality numbers (RQN) above 8.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Barcodes corresponding to nuclei were distinguished from empty droplets using ArchR (1.0.2), requiring at least 5,000 fragments and a minimum TSS enrichment of 3. Doublets were removed through an iterative approach. ArchR (1.0.2) was used to generate the LSI embedding, identify peaks in a cluster-specific and replicate-aware manner (also using MACS2 (2.1.2), estimate gene scores and TF motif accessibility scores (also using chromVar 1.20.2).Sequence Alignment - Raw sequencing data were demultiplexed and converted to fastq format using cellranger-atac mkfastq (1.1.0). Cellranger-atac count (1.1.0) was used to correct droplet barcodes for sequencing errors, align reads to the marmoset genome (CalJac4), generate position-corrected tabular fragment files and identify PCR duplicates from fragments with identical positions originating from the same droplet barcode.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsChromium 10xNextSeq 550Sex-determining pathways produce dimorphic gonads (ovaries and testes), yet the gene regulatory programs governing gonadogenesis and their evolution in primates remain little explored. Here we report evolutionary analyses of transcriptome and chromatin accessibility data of male and female human, marmoset (New World monkey), and mouse gonadal cells spanning key prenatal stages. We find that the two primates and mouse share similar X chromosome expression dynamics, including X chromosome reactivation (XCR), and that in Klinefelter syndrome (XXY) testes, germ cells undergo female-like XCR and escape of X inactivation. New male-specific regulatory regions have emerged progressively during mammalian evolution, especially on the X following sex chromosome origination. Further analyses revealed single nucleus RNA sequencingGallus gallusThe evolution of gene regulatory programs controlling gonadal development in primatesNils Jonathan TrostNils Trost, Amir Fallahshahroudi, Ioannis Sarropoulos, Céline Schneider, Julia Schmidt, Noe Mbengue, Eva Wolff, Charis Drummer, Robert Frömel, Steven Lisgo, Florent Murat, Mari Sepp, Margarida Cardoso-Moreira, Rüdiger Behr, Henrik KaessmannfalsesnRNA-seq of the developing chicken ovaries from E17-E20This study used droplet-based snRNA-seq and joint snRNA-seq and snATAC-seq multiome to profile the gene expression of single nuclei in developing chicken ovaries across three timepoints, from embryonic day (E) 17 to E20. Fertilized chicken eggs (Gallus gallus) were purchased from Granja Santa Isabel and incubated at 37.5 °C in a humidified atmosphere until the required developmental stage. The day when eggs were incubated was considered E0. Single-cell barcoding and library preparation were performed using Chromium Single Cell 3' Reagent Kits (v3 chemistry) for snRNA-seq only and Chromium Single Cell Multiome ATAC + Gene Expression Reagent kits (v1) for multiome experiments with the Chromium Controller instrument (10x Genomics). Sequencing of the libraries was carried out on the Illumina N2026-08-31T00:00:00Z2026-08-31T01:00:47.33Z2026-08-13T20:05:43.614ZE-MTAB-17505ERP203681EFO_0002944EFO_0004170EFO_0004917EFO_0009809EFO_0005518EFO_0003816EFO_000418410.1101/2025.06.17.659946