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The insufficient understanding of their molecular diversity and evolutionary processes hinders the advancement of new treatments. Technical complexities associated with formalin\u2010fixed paraffin\u2010embedded (FFPE) clinical samples hinder molecular\u2010level analyses of gliomas. Current single\u2010cell RNA sequencing (scRNA\u2010seq) platforms are inadequate for large\u2010scale clinical applications. In this study, automated snRandom\u2010seq is developed, a high\u2010throughput single\u2010nucleus total RNA sequencing platform optimized for archival FFPE samples. This platform integrates automated single\u2010nucleus isolation and droplet barcoding systems with the random primer\u2010based scRNA\u2010seq chemistry, accommodating a broad spectrum of sample types. The automated snRandom\u2010seq is applied to analyze 116\u00a0492 single nuclei from 17 FFPE samples of various glioma subtypes, including rare clinical samples and matched primary\u2010recurrent glioblastomas (GBMs). The study provides comprehensive insights into the molecular characteristics of gliomas at the single\u2010cell level. Abundant non\u2010coding RNAs (ncRNAs) with distinct expression profiles across different glioma clusters and uncovered promising recurrence\u2010related targets and pathways in primary\u2010recurrent GBMs are identified. These findings establish automated snRandom\u2010seq as a robust tool for scRNA\u2010seq of FFPE samples, enabling exploration of molecular diversities and tumor evolution. This platform holds significant implications for large\u2010scale integrative and retrospective clinical research.<\/jats:p>","DOI":"10.1002\/smtd.202301801","type":"journal-article","created":{"date-parts":[[2024,7,3]],"date-time":"2024-07-03T06:01:50Z","timestamp":1719986510000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Single Nucleus Total RNA Sequencing of Formalin\u2010Fixed Paraffin\u2010Embedded Gliomas"],"prefix":"10.1002","author":[{"given":"Ziye","family":"Xu","sequence":"first","affiliation":[{"name":"Department of Laboratory Medicine of The First Affiliated Hospital &amp; Liangzhu Laboratory Zhejiang University School of Medicine Hangzhou 310003 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lingchao","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery Huashan Hospital Fudan University Shanghai 200040 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xin","family":"Lin","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine of The First Affiliated Hospital &amp; Liangzhu Laboratory Zhejiang University School of Medicine Hangzhou 310003 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuexiao","family":"Lyu","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine of The First Affiliated Hospital &amp; Liangzhu Laboratory Zhejiang University School of Medicine Hangzhou 310003 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mofei","family":"Zhou","sequence":"additional","affiliation":[{"name":"M20 Genomics Hangzhou 311121 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haide","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine of The First Affiliated Hospital &amp; Liangzhu Laboratory Zhejiang University School of Medicine Hangzhou 310003 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heng","family":"Zhang","sequence":"additional","affiliation":[{"name":"M20 Genomics Hangzhou 311121 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tianyu","family":"Zhang","sequence":"additional","affiliation":[{"name":"M20 Genomics Hangzhou 311121 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine of The First Affiliated Hospital &amp; Liangzhu Laboratory Zhejiang University School of Medicine Hangzhou 310003 China"},{"name":"Zhejiang Key Laboratory of Clinical In Vitro Diagnostic Techniques Hangzhou 310003 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuanzhen","family":"Suo","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine of The First Affiliated Hospital &amp; Liangzhu Laboratory Zhejiang University School of Medicine Hangzhou 310003 China"},{"name":"Jiangsu Healthy Life Innovation Medical Technology Co., Ltd Wuxi 214174 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qian","family":"Liang","sequence":"additional","affiliation":[{"name":"M20 Genomics Hangzhou 311121 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhiyong","family":"Qin","sequence":"additional","affiliation":[{"name":"Department of Neurosurgery Huashan Hospital Fudan University Shanghai 200040 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2820-4243","authenticated-orcid":false,"given":"Yongcheng","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Laboratory Medicine of The First Affiliated Hospital &amp; Liangzhu Laboratory Zhejiang University School of Medicine Hangzhou 310003 China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2024,7,3]]},"reference":[{"key":"e_1_2_9_1_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cancergen.2012.10.009"},{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1093\/neuonc\/nou087"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1001\/jama.2023.0023"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature12625"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2023.07.024"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jmoldx.2011.06.007"},{"key":"e_1_2_9_7_1","unstructured":"F. 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