biostudies-literatureUnknown16(1)Jin WSingle-cell ATAC-seq (scATAC-seq) enables high-resolution mapping of chromatin accessibility but is often limited by throughput, cost, and equipment requirements. Here, we present indexed Tn5 tagmentation-based scATAC-seq (IT-scATAC-seq), a semi-automated, cost-effective, and scalable approach that leverages indexed Tn5 transposomes and a three-round barcoding strategy. This workflow prepares libraries for up to 10,000 cells in a single day, reduces the per-cell cost to approximately $0.01, and maintains high data quality. Comprehensive benchmarking demonstrates that IT-scATAC-seq achieves robust library complexity, high signal specificity, and improved cost-efficiency compared to existing methods. We apply IT-scATAC-seq to mouse embryonic stem cells, capturing chromatin remodelling duringNature communications2635https://www.ebi.ac.uk/biostudies/studies/S-EPMC11914533biostudies-literatureSemi-automated IT-scATAC-seq profiles cell-specific chromatin accessibility in differentiation and peripheral blood populations.PMC11914533Jin WHuang SJin GLi TMa JRong LZhou ZfalseSemi-automated IT-scATAC-seq profiles cell-specific chromatin accessibility in differentiation and peripheral blood populations.Single-cell ATAC-seq (scATAC-seq) enables high-resolution mapping of chromatin accessibility but is often limited by throughput, cost, and equipment requirements. Here, we present indexed Tn5 tagmentation-based scATAC-seq (IT-scATAC-seq), a semi-automated, cost-effective, and scalable approach that leverages indexed Tn5 transposomes and a three-round barcoding strategy. This workflow prepares libraries for up to 10,000 cells in a single day, reduces the per-cell cost to approximately $0.01, and maintains high data quality. Comprehensive benchmarking demonstrates that IT-scATAC-seq achieves robust library complexity, high signal specificity, and improved cost-efficiency compared to existing methods. We apply IT-scATAC-seq to mouse embryonic stem cells, capturing chromatin remodelling during2025-01-01T00:00:00Z2025 Mar2026-06-01T06:07:08.579Z2025-04-06T22:09:52.236ZS-EPMC119145334009744410.1038/s41467-025-57931-2