single cell chromatin accessiblity profiling in formlin fixed paraffin embedded samples
We evaluated the performance of ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) on formalin-fixed paraffin-embedded (FFPE) spleen samples from C57BL/6J mice. Nuclei were isolated from FFPE mouse spleen tissue, and ATAC-seq was performed at both the bulk and single-cell levels, with and without reverse crosslinking. The resulting datasets were compared to ATAC-seq and scATAC-seq profiles obtained from fresh spleen samples. Our analysis revealed a markedly reduced library complexity in FFPE-derived samples compared to fresh tissue, across both bulk and single-cell experiments. These findings highlight the technical limitations of applying standard ATAC-seq protocols to FFPE samples. Based on our results, we conclude that conventional ATAC-seq approaches are not well-suited for chromatin accessibility profiling in FFPE-preserved tissues. Overall design: Nuclei were isolated from snap-frozen FFPE spleen tissues of C57BL/6J mice following the Omni-ATAC-seq protocol. A total of 20,000 to 100,000 DAPI-positive nuclei were counted and collected. The nuclei were pelleted and resuspended in 1 mL of ATAC-Resuspension Buffer (ATAC-RSB) supplemented with 0.1% Tween-20. Transposition was performed using Tn5 transposase. The tagmented DNA was purified under both reverse crosslinking and non-crosslinking conditions, and subsequently PCR-amplified for 8–15 cycles using indexed primers to introduce sample-specific barcodes. For single-cell ATAC-seq, a combinatorial indexing (split-pool) strategy was employed to profile chromatin accessibility in individual nuclei isolated from FFPE mouse spleen samples. Nuclei were first isolated using a modified Omni-ATAC protocol optimized for FFPE tissue. After DAPI staining and counting, the nuclei were subjected to a three-level barcoding scheme. In the first round, transposition was performed using indexed Tn5 transposase. The nuclei were then split across multiple wells and underwent successive rounds of PCR with unique barcoded primers at each step. This split-pool barcoding enabled the unique indexing of individual nuclei. Libraries were pooled, purified under both reverse crosslinking and non-crosslinking conditions, and sequenced to generate single-nucleus ATAC-seq data.