{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,30]],"date-time":"2026-10-30T07:51:56Z","timestamp":1793346716840,"version":"4.5.0"},"posted":{"date-parts":[[2025,3,26]]},"group-title":"Neuroscience","reference-count":63,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,3,26]],"date-time":"2025-03-26T00:00:00Z","timestamp":1742947200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,3,26]]},"abstract":"<jats:title>SUMMARY<\/jats:title>\n                <jats:p>Single-nucleus RNA sequencing (snRNA-seq) enables resolving cellular heterogeneity in complex tissues. snRNA-seq overcomes limitations of traditional single-cell RNA-seq by using nuclei instead of cells, allowing to utilize frozen tissues and difficult-to-isolate cell types. Although various nuclei isolation methods have been developed, systematic evaluations of their effects on nuclear integrity and subsequent data quality remain lacking, a critical gap with profound implications for the rigor and reproducibility. To address this, we compared three mechanistically distinct nuclei isolation strategies with brain tissues: a sucrose gradient centrifugation-based method, a spin column-based method, and a machine-assisted platform. All methods successfully captured diverse cell types but revealed considerable protocol-dependent differences in cell type proportions, transcriptional homogeneity, and the preservation of cell-type-specific and cell-state-specific markers. Moreover, isolation workflows differentially influenced contamination levels from ambient, mitochondrial, and ribosomal RNAs. Our findings establish nuclei isolation methodology as a critical experimental variable shaping snRNA-seq data quality and biological interpretation.<\/jats:p>\n                <jats:sec>\n                  <jats:title>MOTIVATION<\/jats:title>\n                  <jats:p>Single-nucleus RNA sequencing (snRNA-seq) has become an essential tool for transcriptomic analysis of complex tissues. However, the quality and efficiency of data generation depend heavily on the method used for nuclear isolation. The existing isolation techniques vary in their ability to preserve nuclear integrity, minimize ambient RNA contamination, and optimize recovery rates. Despite these differences in quality, a systematic comparison of these methods, specifically for brain tissue, is lacking. This gap poses a challenge for researchers in choosing the most suitable approach for their particular experimental requirements. To address this critical issue, our study directly compared three nuclei isolation methods and evaluated their performance in terms of yield, purity, and downstream sequencing quality. By providing a comprehensive assessment, we aim to guide researchers in selecting the most appropriate isolation protocol for their snRNA-seq experiments, ensuring optimal results and advancing the study of complex brain tissues at the single-nucleus level.<\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.03.25.645306","type":"posted-content","created":{"date-parts":[[2025,3,27]],"date-time":"2025-03-27T03:09:10Z","timestamp":1743044950000},"source":"Crossref","is-referenced-by-count":4,"title":["Comparative analysis of nuclei isolation methods for brain single-nucleus RNA sequencing"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2168-6139","authenticated-orcid":false,"given":"Holly N.","family":"Kersey","sequence":"first","affiliation":[{"name":"Medical Neurosciences Graduate Program, Indiana University School of Medicine, Indianapolis, IN, USA"},{"name":"Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dominic J.","family":"Acri","sequence":"additional","affiliation":[{"name":"Medical Neurosciences Graduate Program, Indiana University School of Medicine, Indianapolis, IN, USA"},{"name":"Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luke C.","family":"Dabin","sequence":"additional","affiliation":[{"name":"Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, USA"},{"name":"Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kelly","family":"Hartigan","sequence":"additional","affiliation":[{"name":"Medical Neurosciences Graduate Program, Indiana University School of Medicine, Indianapolis, IN, USA"},{"name":"Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richard","family":"Mustaklem","sequence":"additional","affiliation":[{"name":"Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, USA"},{"name":"Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jung Hyun","family":"Park","sequence":"additional","affiliation":[{"name":"Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, USA"},{"name":"Department of Psychological and Brain Sciences, Indiana University, Bloomington, IN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6931-8581","authenticated-orcid":false,"given":"Jungsu","family":"Kim","sequence":"additional","affiliation":[{"name":"Stark Neurosciences Research Institute, Indiana University School of Medicine, Indianapolis, IN, USA"},{"name":"Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026041201034288000_2025.03.25.645306v1.1","doi-asserted-by":"crossref","unstructured":"Macnair, W. , D. 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