Enhancing single-cell ATAC sequencing with formaldehyde fixation, cryopreservation, and multiplexing for flexible...

Area of Science:

• Molecular Biology
• Genomics
• Epigenetics

Background:

• Freshly isolated cells are required for bulk and single-cell ATAC-seq, posing logistical challenges and increasing batch effects.
• Coordinating complex or longitudinal studies is difficult due to the need for immediate sample processing.

Purpose of the Study:

• To develop a sample preservation strategy for consistent, high-quality chromatin accessibility profiling from archived samples.
• To overcome limitations associated with fresh sample requirements in ATAC-seq experiments.

Main Methods:

• A workflow involving mild formaldehyde fixation followed by cryopreservation was established.
• The protocol was validated for both bulk and single-cell ATAC-seq using HepG2 cells.
• Compatibility with transposase-based sample multiplexing and a computational demultiplexing strategy were developed.

Main Results:

• The preservation method maintained ATAC-seq data quality comparable to fresh samples.
• Key data quality metrics, including signal-to-noise ratio and fragment distributions, were reliably preserved.
• The computational demultiplexing strategy accurately assigned single cells to their sample of origin, addressing barcode hopping.

Conclusions:

• This approach streamlines experimental logistics and enhances reproducibility for ATAC-seq studies.
• The method enables high-quality chromatin accessibility profiling from archived samples, broadening study scope.
• The strategy is particularly beneficial for clinical research settings with challenging sample collection logistics.