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Diverse single\u2010nucleus RNA sequencing (snRNA\u2010seq) techniques have been developed for FFPE samples, however, how to perform high\u2010throughput snRNA\u2010seq on small FFPE puncture samples is still a challenge. Here, the previously developed snRNA\u2010seq technique (snRandom\u2010seq) is optimized by implementing a pre\u2010indexing procedure for the minimal puncture FFPE samples. In analyzing 20 samples from various solid tumors, optimized snRandom\u2010seq still detected \u224817\u00a0000 genes and 12\u00a0000 long non\u2010coding RNAs (lncRNAs), achieving precise clustering based on tissue origin. A head\u2010to\u2010head comparison with 10\u00d7 Genomics on fresh biopsy samples showed a similar gene detection rate, with significantly enhanced lncRNA detection, indicating that the optimized snRandom\u2010seq technique maintains its established gene detection advantages even when applied to small samples. Utilizing 7 puncture FFPE samples of liver metastases from 3 colorectal cancer patients pre\u2010 and post\u2010immunotherapy, the cellular developmental trajectories are reconstructed and revealed dynamic spatiotemporal heterogeneity during treatment, including insights into pseudoprogression of immunotherapy. Therefore, the optimized snRandom\u2010seq offers a solution for high\u2010throughput single\u2010cell RNA and non\u2010coding RNA analysis in minimal puncture FFPE sample.<\/jats:p>","DOI":"10.1002\/advs.202410713","type":"journal-article","created":{"date-parts":[[2024,12,4]],"date-time":"2024-12-04T10:11:24Z","timestamp":1733307084000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["High\u2010Throughput Single\u2010Nucleus RNA Profiling of Minimal Puncture FFPE Samples Reveals Spatiotemporal Heterogeneity of Cancer"],"prefix":"10.1002","volume":"12","author":[{"given":"Weiqin","family":"Jiang","sequence":"first","affiliation":[{"name":"Department of Colorectal Surgery the First Affiliated Hospital Zhejiang University School of Medicine  Hangzhou 310003 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiang","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Colorectal Surgery the First Affiliated Hospital Zhejiang University School of Medicine  Hangzhou 310003 China"},{"name":"The First Clinical Medical College of Lanzhou University  Lanzhou 730000 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ziye","family":"Xu","sequence":"additional","affiliation":[{"name":"Liangzhu Laboratory Zhejiang University Medical Center  Hangzhou 311121 China"},{"name":"Department of Laboratory Medicine the First Affiliated Hospital Zhejiang University School of Medicine  Hangzhou 311121 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qing","family":"Cheng","sequence":"additional","affiliation":[{"name":"Institute of Bioinformatics and James D. Watson Institute of Genome Sciences Zhejiang University  Hangzhou 310058 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaohan","family":"Li","sequence":"additional","affiliation":[{"name":"Institute of Bioinformatics and James D. Watson Institute of Genome Sciences Zhejiang University  Hangzhou 310058 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuyi","family":"Zhu","sequence":"additional","affiliation":[{"name":"Liangzhu Laboratory Zhejiang University Medical Center  Hangzhou 311121 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fangru","family":"Lu","sequence":"additional","affiliation":[{"name":"Liangzhu Laboratory Zhejiang University Medical Center  Hangzhou 311121 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ling","family":"Dong","sequence":"additional","affiliation":[{"name":"M20 Genomics  Hangzhou 310030 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Linghui","family":"Zeng","sequence":"additional","affiliation":[{"name":"School of Medicine Hangzhou City University  Hangzhou 316021 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weixiang","family":"Zhong","sequence":"additional","affiliation":[{"name":"Department of Pathology First Affiliated Hospital College of Medicine Zhejiang University  Hangzhou 310003 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yongcheng","family":"Wang","sequence":"additional","affiliation":[{"name":"Liangzhu Laboratory Zhejiang University Medical Center  Hangzhou 311121 China"},{"name":"Department of Laboratory Medicine the First Affiliated Hospital Zhejiang University School of Medicine  Hangzhou 311121 China"},{"name":"College of Biomedical Engineering and Instrument Science Zhejiang University  Hangzhou 310027 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Longjiang","family":"Fan","sequence":"additional","affiliation":[{"name":"Institute of Bioinformatics and James D. Watson Institute of Genome Sciences Zhejiang University  Hangzhou 310058 China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5980-1262","authenticated-orcid":false,"given":"Hongyu","family":"Chen","sequence":"additional","affiliation":[{"name":"School of Medicine Hangzhou City University  Hangzhou 316021 China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2024,12,4]]},"reference":[{"key":"e_1_2_9_1_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-22801-0"},{"key":"e_1_2_9_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-34395-2"},{"key":"e_1_2_9_3_1","doi-asserted-by":"publisher","DOI":"10.1186\/s13073-023-01164-9"},{"key":"e_1_2_9_4_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2103240118"},{"key":"e_1_2_9_5_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-2756-3_19"},{"key":"e_1_2_9_6_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41568-019-0180-2"},{"key":"e_1_2_9_7_1","doi-asserted-by":"publisher","DOI":"10.1080\/10520295.2018.1446101"},{"key":"e_1_2_9_8_1","doi-asserted-by":"publisher","DOI":"10.2214\/ajr.142.1.41"},{"key":"e_1_2_9_9_1","volume-title":"Percutaneous Liver Biopsy","author":"Chan M.","year":"2023"},{"key":"e_1_2_9_10_1","doi-asserted-by":"publisher","DOI":"10.1148\/radiol.2019182584"},{"key":"e_1_2_9_11_1","doi-asserted-by":"publisher","DOI":"10.1158\/0008-5472.CAN-18-3402"},{"key":"e_1_2_9_12_1","doi-asserted-by":"publisher","DOI":"10.1186\/s12967-019-1864-9"},{"key":"e_1_2_9_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.clinbiochem.2013.12.011"},{"key":"e_1_2_9_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.xgen.2021.100065"},{"key":"e_1_2_9_15_1","author":"Chung H.","year":"2022","journal-title":"bioRxiv"},{"key":"e_1_2_9_16_1","author":"Vallejo A. 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