{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T10:31:22Z","timestamp":1791973882839,"version":"4.4.0"},"reference-count":18,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2018,2,1]],"date-time":"2018-02-01T00:00:00Z","timestamp":1517443200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2018,2,1]],"date-time":"2018-02-01T00:00:00Z","timestamp":1517443200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>The design and implementation of single-cell experiments is often limited by their requirement for fresh starting material. We have adapted a method for histological tissue fixation using dithio-bis(succinimidyl propionate) (DSP), or Lomant\u2019s Reagent, to stabilise cell samples for single-cell transcriptomic applications. DSP is a reversible cross-linker of free amine groups that has previously been shown to preserve tissue integrity for histology while maintaining RNA integrity and yield in bulk RNA extractions. Although RNA-seq data from DSP-fixed single cells appears to be prone to characteristic artefacts, such as slightly reduced yield of cDNA and a detectable 3\u2032 bias in comparison with fresh cells, cell preservation using DSP does not appear to substantially reduce RNA complexity at the gene level. In addition, there is evidence that instantaneous fixation of cells can reduce inter-cell technical variability. The ability of DSP-fixed cells to retain commonly used dyes, such as propidium iodide, enables the tracking of experimental sub-populations and the recording of cell viability at the point of fixation. Preserving cells using DSP will remove several barriers in the staging of single-cell experiments, including the transport of samples and the scheduling of shared equipment for downstream single-cell isolation and processing.<\/jats:p>","DOI":"10.1038\/s41598-018-20372-7","type":"journal-article","created":{"date-parts":[[2018,1,26]],"date-time":"2018-01-26T09:31:27Z","timestamp":1516959087000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":90,"title":["A practical solution for preserving single cells for RNA sequencing"],"prefix":"10.1038","volume":"8","author":[{"given":"Moustafa","family":"Attar","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eshita","family":"Sharma","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuqiang","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Claire","family":"Bryer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura","family":"Cubitt","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John","family":"Broxholme","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Helen","family":"Lockstone","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James","family":"Kinchen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alison","family":"Simmons","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paolo","family":"Piazza","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Buck","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9105-5856","authenticated-orcid":false,"given":"Kenneth J.","family":"Livak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rory","family":"Bowden","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2018,2,1]]},"reference":[{"key":"20372_CR1","doi-asserted-by":"publisher","first-page":"41","DOI":"10.1038\/nmeth.2694","volume":"11","author":"AR Wu","year":"2014","unstructured":"Wu, A. R. et al. Quantitative assessment of single-cell RNA-sequencing methods. Nat Methods 11, 41\u201346, https:\/\/doi.org\/10.1038\/nmeth.2694 (2014).","journal-title":"Nat Methods"},{"key":"20372_CR2","doi-asserted-by":"publisher","first-page":"94","DOI":"10.1002\/cyto.a.22352","volume":"85","author":"N Beliakova-Bethell","year":"2014","unstructured":"Beliakova-Bethell, N. et al. The effect of cell subset isolation method on gene expression in leukocytes. Cytometry A 85, 94\u2013104, https:\/\/doi.org\/10.1002\/cyto.a.22352 (2014).","journal-title":"Cytometry A"},{"key":"20372_CR3","doi-asserted-by":"publisher","first-page":"87","DOI":"10.1038\/nmeth.3629","volume":"13","author":"ER Thomsen","year":"2016","unstructured":"Thomsen, E. R. et al. Fixed single-cell transcriptomic characterization of human radial glial diversity. Nat Methods 13, 87\u201393, https:\/\/doi.org\/10.1038\/nmeth.3629 (2016).","journal-title":"Nat Methods"},{"key":"20372_CR4","doi-asserted-by":"publisher","DOI":"10.1186\/s13059-017-1171-9","volume":"18","author":"A Guillaumet-Adkins","year":"2017","unstructured":"Guillaumet-Adkins, A. et al. Single-cell transcriptome conservation in cryopreserved cells and tissues. Genome Biol 18, 45, https:\/\/doi.org\/10.1186\/s13059-017-1171-9 (2017).","journal-title":"Genome Biol"},{"key":"20372_CR5","doi-asserted-by":"publisher","DOI":"10.1186\/s12915-017-0383-5","volume":"15","author":"J Alles","year":"2017","unstructured":"Alles, J. et al. Cell fixation and preservation for droplet-based single-cell transcriptomics. BMC Biol 15, 44, https:\/\/doi.org\/10.1186\/s12915-017-0383-5 (2017).","journal-title":"BMC Biol"},{"key":"20372_CR6","doi-asserted-by":"publisher","first-page":"e185","DOI":"10.1093\/nar\/gnh185","volume":"32","author":"CC Xiang","year":"2004","unstructured":"Xiang, C. C. et al. Using DSP, a reversible cross-linker, to fix tissue sections for immunostaining, microdissection and expression profiling. Nucleic Acids Res 32, e185, https:\/\/doi.org\/10.1093\/nar\/gnh185 (2004).","journal-title":"Nucleic Acids Res"},{"key":"20372_CR7","doi-asserted-by":"publisher","DOI":"10.1186\/1472-6750-13-104","volume":"13","author":"S Lamble","year":"2013","unstructured":"Lamble, S. et al. Improved workflows for high throughput library preparation using the transposome-based Nextera system. BMC Biotechnol 13, 104, https:\/\/doi.org\/10.1186\/1472-6750-13-104 (2013).","journal-title":"BMC Biotechnol"},{"key":"20372_CR8","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2105-15-182","volume":"15","author":"H Jiang","year":"2014","unstructured":"Jiang, H., Lei, R., Ding, S. W. & Zhu, S. Skewer: a fast and accurate adapter trimmer for next-generation sequencing paired-end reads. BMC Bioinformatics 15, 182, https:\/\/doi.org\/10.1186\/1471-2105-15-182 (2014).","journal-title":"BMC Bioinformatics"},{"key":"20372_CR9","doi-asserted-by":"publisher","first-page":"357","DOI":"10.1038\/nmeth.3317","volume":"12","author":"D Kim","year":"2015","unstructured":"Kim, D., Langmead, B. & Salzberg, S. L. HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12, 357\u2013360, https:\/\/doi.org\/10.1038\/nmeth.3317 (2015).","journal-title":"Nat Methods"},{"key":"20372_CR10","doi-asserted-by":"publisher","first-page":"2078","DOI":"10.1093\/bioinformatics\/btp352","volume":"25","author":"H Li","year":"2009","unstructured":"Li, H. et al. The Sequence Alignment\/Map format and SAMtools. Bioinformatics 25, 2078\u20132079, https:\/\/doi.org\/10.1093\/bioinformatics\/btp352 (2009).","journal-title":"Bioinformatics"},{"key":"20372_CR11","doi-asserted-by":"publisher","first-page":"D749","DOI":"10.1093\/nar\/gkt1196","volume":"42","author":"P Flicek","year":"2014","unstructured":"Flicek, P. et al. Ensembl 2014. Nucleic Acids Res 42, D749\u2013755, https:\/\/doi.org\/10.1093\/nar\/gkt1196 (2014).","journal-title":"Nucleic Acids Res"},{"key":"20372_CR12","doi-asserted-by":"publisher","first-page":"923","DOI":"10.1093\/bioinformatics\/btt656","volume":"30","author":"Y Liao","year":"2014","unstructured":"Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923\u2013930, https:\/\/doi.org\/10.1093\/bioinformatics\/btt656 (2014).","journal-title":"Bioinformatics"},{"key":"20372_CR13","doi-asserted-by":"publisher","first-page":"e108","DOI":"10.1093\/nar\/gkt214","volume":"41","author":"Y Liao","year":"2013","unstructured":"Liao, Y., Smyth, G. K. & Shi, W. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res 41, e108, https:\/\/doi.org\/10.1093\/nar\/gkt214 (2013).","journal-title":"Nucleic Acids Res"},{"key":"20372_CR14","unstructured":"R Core Team. R: A language and environment for statistical computing,. Vienna: R Foundation for Statistical Computing, Vienna, Austria.; 2007."},{"key":"20372_CR15","doi-asserted-by":"publisher","first-page":"483","DOI":"10.1038\/nmeth.4236","volume":"14","author":"VY Kiselev","year":"2017","unstructured":"Kiselev, V. Y. et al. SC3: consensus clustering of single-cell RNA-seq data. Nat Methods 14, 483\u2013486, https:\/\/doi.org\/10.1038\/nmeth.4236 (2017).","journal-title":"Nat Methods"},{"key":"20372_CR16","doi-asserted-by":"publisher","first-page":"e23780","DOI":"10.1371\/journal.pone.0023780","volume":"6","author":"C Mueller","year":"2011","unstructured":"Mueller, C. et al. One-step preservation of phosphoproteins and tissue morphology at room temperature for diagnostic and research specimens. PLoS One 6, e23780, https:\/\/doi.org\/10.1371\/journal.pone.0023780 (2011).","journal-title":"PLoS One"},{"key":"20372_CR17","unstructured":"Espina VA, L. L., Mueller C. One-Step Cell and Tissue Preservative for Morphologic and Molecular Analysis (2013)."},{"key":"20372_CR18","doi-asserted-by":"publisher","first-page":"207","DOI":"10.1093\/nar\/30.1.207","volume":"30","author":"R Edgar","year":"2002","unstructured":"Edgar, R., Domrachev, M. & Lash, A. E. Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 30, 207\u2013210 (2002).","journal-title":"Nucleic Acids Res"}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-018-20372-7","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-018-20372-7.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-018-20372-7.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,20]],"date-time":"2022-12-20T20:51:06Z","timestamp":1671569466000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-018-20372-7"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,2,1]]},"references-count":18,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2018,12]]}},"alternative-id":["20372"],"URL":"https:\/\/doi.org\/10.1038\/s41598-018-20372-7","relation":{"has-preprint":[{"id-type":"doi","id":"10.1101\/160804","asserted-by":"object"}]},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2018,2,1]]},"assertion":[{"value":"20 September 2017","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"4 January 2018","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"1 February 2018","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"K.J.L. is a former employee of Fluidigm.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing Interests"}}],"article-number":"2151"}}