@article{HohlBoenischMankeetal., author = {Hohl, Tobias and B{\"o}nisch, Ulrike and Manke, Thomas and Arrigoni, Laura}, title = {Enhancing single-cell ATAC sequencing with formaldehyde fixation, cryopreservation, and multiplexing for flexible analysis}, series = {BMC Research Notes}, volume = {18}, journal = {BMC Research Notes}, number = {1}, publisher = {Springer Nature}, address = {London}, issn = {1756-0500}, doi = {10.1186/s13104-025-07547-y}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:523-21739}, abstract = {Objective: The need for freshly isolated cells in bulk or single cell ATAC-seq experiments creates considerable logistical barriers and increases susceptibility to batch effects. This makes it difficult to coordinate complex or longitudinal studies. Our goal was to develop a sample preservation strategy that overcomes these limitations, enabling consistent and high-quality chromatin accessibility profiling from archived samples. Results: We established a workflow that incorporates mild formaldehyde fixation prior to cryopreservation, preserving both bulk and single-cell ATAC-seq data quality at levels comparable to fresh samples in HepG2 cells. This protocol reliably maintains key data quality metrics, including signal-to-noise ratio and fragment distributions. Furthermore, the method is fully compatible with transposase-based sample multiplexing using custom Tn5 barcodes. To address barcode hopping inherent to multiplexing, we introduced a computational demultiplexing strategy based on fragment ratios, which accurately assigns single cells to their sample of origin. Our approach streamlines experimental logistics and ensures reproducibility across diverse and temporally dispersed samples, broadening the scope for ATAC-seq-based studies, including those in clinical research settings where coordinated sample collection is challenging.}, subject = {Single Cell Analysis}, language = {en} }