An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide-conjugated antibodies

Area of Science:

• Genomics and Molecular Biology
• Epigenetics and Chromatin Biology
• Single-Cell Analysis Technologies

Background:

• Single-cell technologies are crucial for understanding cellular functions and molecular properties.
• Sample multiplexing in single-cell analysis improves efficiency and reduces technical variation.
• Multiplexing techniques for single nuclear assay for transposase-accessible chromatin (snATAC)-seq are still developing.

Purpose of the Study:

• To develop a simple and effective nucleus hashing method for multiplexing snATAC-seq.
• To enable high-accuracy sample demultiplexing in snATAC-seq experiments.
• To facilitate cost-efficient and technically robust snATAC-seq library preparation.

Main Methods:

• Developed NuHash, a nucleus hashing method using oligonucleotide-conjugated antibodies against nuclear pore complex proteins.
• Applied NuHash for multiplexing snATAC-seq on mixtures of human and mouse cell samples (2-plex and 4-plex).
• Validated demultiplexing accuracy by comparing NuHash results with reference genome alignments.

Main Results:

• NuHash achieved high demultiplexing accuracy in both 2-plex and 4-plex snATAC-seq experiments.
• Differential open chromatin region (OCR) analysis identified sex-specific OCRs on chromosomes X and Y.
• Bulk ATAC-seq signal intensity correlated positively with the number of cell clusters detected in snATAC-seq, suggesting its utility in identifying functional loci.

Conclusions:

• NuHash is a straightforward and effective method for high-accuracy sample multiplexing in snATAC-seq.
• The NuHash protocol is compatible with frozen samples and requires no modifications to standard snATAC-seq library preparation.
• This method significantly advances the capabilities of snATAC-seq for large-scale and cost-efficient chromatin accessibility studies.