scFFPE-ATAC enables high-throughput single cell chromatin accessibility profiling in formalin-fixed paraffin-embedded samples.

Formalin-fixed paraffin-embedded (FFPE) samples are the gold standard for tissue preservation in clinical and research settings. Current single-cell chromatin accessibility technologies cannot resolve cell-type-specific epigenetic profiles in FFPE tissues due to extensive DNA damage. We present scFFPE-ATAC, a high-throughput single-cell chromatin accessibility assay for FFPE samples that integrates an FFPE-adapted Tn5 transposase, ultra-high-throughput DNA barcoding (>56 million barcodes per run), T7 promoter-mediated DNA damage repair, and in vitro transcription. We benchmark scFFPE-ATAC on FFPE mouse spleen and validate its performance against fresh tissue. We apply it to human lymph node samples archived for 8-12 years and to lung cancer FFPE tissues, revealing distinct regulatory trajectories between tumor center and invasive edge. Analysis of archived follicular lymphoma and transformed diffuse large B-cell lymphoma samples identifies relapse- and transformation-associated epigenetic dynamics. scFFPE-ATAC enables retrospective, spatial, and mechanistic epigenetic studies in long-term archived specimens.