biostudies-arrayexpressNils Jonathan TrostGallus galluscellranger-arc (2.0.2)ArchR (1.0.2), MACS2 (2.1.2), chromVar (1.20.2)https://www.ebi.ac.uk/biostudies/studies/E-MTAB-17506This study used droplet-based 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 E19. 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 Multiome ATAC + Gene Expression Reagent kits (v1) with the Chromium Controller instrument (10x Genomics). Sequencing of the libraries was carried out on the Illumina NextSeq 500/550 and initial data processing was performed using Cellranger.biostudies-arrayexpressSequencing - Sequencing was performed on Illumina NextSeq 550 (RRID:SCR_016381). For the ATAC modality, sequencing was run with 50 cycles for both Read 1 and Read 2, 8 cycles for the i7 index, and 16 cycles for the i5 index. For the RNA modality, sequencing was run with 28 cycles for Read 1, 90 cycles for Read 2, and 10 cycles for the indices.Library Construction - Nuclei were processed using Chromium Single Cell Multiome ATAC + Gene Expression Reagent kits (v1) and the Chromium Controller instrument (10x Genomics; RRID:SCR_019326), following the manufacturer's guidelines. The libraries for the ATAC modality were then amplified through 8 PCR cycles. The libraries for the Gene expression modality were amplified through 7 PCR cycles. They were 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).Nucleic Acid Extraction - 15,000 nuclei per sample were subjected to tagmentation, single cell barcoding and library construction using the Chromium Single Cell ATAC Reagent kits (v1) and Chromium Controller instrument (10X Genomics) according to the manufacturer’s protocols.Sample Collection - rozen tissue was homogenized on ice in a buffer containing 250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl (pH 8), 0.1% IGEPAL, 1 µM DTT, 0.4 U/µl Murine RNase Inhibitor (New England BioLabs), 0.2 U/µl SUPERase-In (Thermo Fisher Scientific). 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 cytoplasmic fraction (supernatant). Nuclei were washed once in the homogenization buffer and resuspended in PBS. Then nuclei were filtered using 40 µm Flowmi strainers. Nuclei concentration was determined by staining with Hoechst DNA dye or propidiOrganizationMINSEQE 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 scATAC-seqGallus 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 KaessmannfalsesnATAC-seq of the developing chicken ovaries from E17-E19This study used droplet-based 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 E19. 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 Multiome ATAC + Gene Expression Reagent kits (v1) with the Chromium Controller instrument (10x Genomics). Sequencing of the libraries was carried out on the Illumina NextSeq 500/550 and initial data processing was performed using Cellranger.2026-08-31T00:00:00Z2026-08-31T01:00:46.8Z2026-08-13T20:26:14.548ZE-MTAB-17506ERP203683EFO_0002944EFO_0004170EFO_0010891EFO_0004917EFO_0005518EFO_0003816EFO_000418410.1101/2025.06.17.659946