{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,8,2]],"date-time":"2025-08-02T19:18:29Z","timestamp":1754162309706,"version":"3.41.2"},"reference-count":28,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T00:00:00Z","timestamp":1753833600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Genet."],"abstract":"<jats:sec><jats:title>Objective<\/jats:title><jats:p>Single-cell transcriptome sequencing is a powerful tool for investigating cellular diversity in normal development and disease. However, prevalent methods predominantly employ 3\u2032-end sequencing of transcripts, limiting the analysis of alternative splicing and other post-transcriptional processes. While full-length single-cell transcriptome sequencing methods, such as Smart-seq, offer more comprehensive information, but are restricted by low-throughput. To overcome these limitations, we propose a strategy that combines <jats:italic>in situ<\/jats:italic> reverse transcription and transposition with a high-throughput micro-fluid platform to enable scalable full-length transcriptome profiling at single-cell resolution.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>In this study, we utilized methanol fixation on cultured cells to evaluate RNA integrity and cellular preservation post-fixation. <jats:italic>In situ<\/jats:italic> reverse transcription followed by RNA\/DNA hybrids transposition was performed to test the efficiency of these reactions. The transposed fragments were sequenced and investigated to determine transcriptome capture efficiency. Finally, we combined <jats:italic>in situ<\/jats:italic> reactions with the 10X Genomics scATAC-seq platform to prepare a single-cell transcriptome library, aiming to assess the feasibility of full-length transcriptome sequencing at the single-cell level using this combined approach.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Methanol fixation enables preservation of RNA and facilitates <jats:italic>in situ<\/jats:italic> reverse transcription of full-length cDNA. Importantly, cells maintain their integrality after reverse transcription and transposition even under low concentration of methanol. Reducing the methanol concentration to 40% further enhances transcript capture efficiency. At the single-cell level, this strategy enables the capture of full-length transcriptomes, demonstrating a great potential for application in single-cell sequencing.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fgene.2025.1629655","type":"journal-article","created":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T04:13:28Z","timestamp":1753848808000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Methanol fixation and tagmentation of RNA\/DNA hybrids directly enable single-cell transcriptome sequencing"],"prefix":"10.3389","volume":"16","author":[{"given":"Tao","family":"Xu","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yicong","family":"Xu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ziyang","family":"An","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yiheng","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jiawen","family":"Yang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Weixing","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jin","family":"Xu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2025,7,30]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"R119","DOI":"10.1186\/gb-2010-11-12-r119","article-title":"Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition","volume":"11","author":"Adey","year":"2010","journal-title":"Genome Biol."},{"key":"B2","doi-asserted-by":"publisher","first-page":"1213","DOI":"10.1038\/nmeth.2688","article-title":"Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position","volume":"10","author":"Buenrostro","year":"2013","journal-title":"Nat. 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