{"doi":"10.1101/2022.12.22.521637","title":"An optimized approach for multiplexing single-nuclear ATAC-seq using oligonucleotide conjugated antibodies","abstract":"Abstract Background Single-cell technologies to analyze transcription and chromatin structure have been widely used in many research areas to reveal the functions and molecular properties of cells at single-cell resolution. Sample multiplexing techniques are valuable when performing single-cell analysis, reducing technical variation and permitting cost efficiencies. Several commercially available methods are available and have been used in many scRNA-seq studies. On the other hand, while several methods have been published, the multiplexing techniques for single nuclear Assay for Transposase-Accessible Chromatin (snATAC)-seq assays remain under development. We developed a simple nucleus hashing method using oligonucleotide conjugated antibodies recognizing nuclear pore complex proteins, NuHash, to perform snATAC-seq library preparations by multiplexing. Results We performed multiplexing snATAC-seq analyses on the mixture of human and mouse cell samples (two samples, 2-plex, and four samples, 4-plex) using NuHash. The demultiplexing accuracy of NuHash was high, and only ten out of 9,144 nuclei (2-plex) and 150 of 12,208 nuclei (4-plex) had discordant classifications between NuHash demultiplexing and discrimination using reference genome alignments. We compared results between snATAC-seq and deeply sequenced bulk ATAC-seq on the same samples and found that most of the peaks detected in snATAC-seq were also detected in deeply sequenced bulk ATAC-seq. The bulk ATAC-seq signal intensity was positively correlated with the number of cell subtype clusters detected in snATAC-seq, but not the subset of peaks detected in all clusters. These subsets of snATAC-seq peaks showed different distributions over different genomic features, suggesting that the peak intensities of bulk ATAC-seq can be used to identify different types of functional loci. Conclusions Our multiplexing method using oligo-conjugated anti-nuclear pore complex proteins, NuHash, permits high accuracy demultiplexing of samples. The NuHash protocol is straightforward, it works on frozen samples, and requires no modifications for snATAC-seq library preparation.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2022,"id":313859,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9523,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":949304,"name":"Taylor V. Thompson","orcid":"0000-0002-0378-0052","position":1,"is_corresponding":false},{"id":467122,"name":"Eric Sosa","orcid":"0000-0001-5965-3477","position":2,"is_corresponding":false},{"id":587444,"name":"Hiroko Nomaru","orcid":null,"position":3,"is_corresponding":false},{"id":1012893,"name":"David M. Reynolds","orcid":null,"position":4,"is_corresponding":false},{"id":78683,"name":"Robert Dubin","orcid":"0000-0001-5556-7248","position":5,"is_corresponding":false},{"id":1012894,"name":"Shahina B. Maqbool","orcid":null,"position":6,"is_corresponding":false},{"id":284103,"name":"Deyou Zheng","orcid":"0000-0003-4354-5337","position":7,"is_corresponding":false},{"id":263880,"name":"Bernice E. Morrow","orcid":"0000-0002-8076-4726","position":8,"is_corresponding":false},{"id":275114,"name":"John M. Greally","orcid":"0000-0001-6069-7960","position":9,"is_corresponding":false},{"id":585984,"name":"Masako Suzuki","orcid":"0000-0003-0605-9225","position":10,"is_corresponding":false},{"id":1012892,"name":"Betelehem Solomon Bera","orcid":null,"position":0,"is_corresponding":true}],"reference_count":33,"raw_metadata":null,"created_at":"2026-07-19T00:33:48.490749Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}