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dc.contributor.authorUniken Venema, Werna TC
dc.contributor.authorRamírez-Sánchez, Aarón D
dc.contributor.authorBigaeva, Emilia
dc.contributor.authorWithoff, Sebo
dc.contributor.authorJonkers, Iris
dc.contributor.authorMcIntyre, Rebecca E
dc.contributor.authorGhouraba, Mennatallah
dc.contributor.authorRaine, Tim
dc.contributor.authorWeersma, Rinse K
dc.contributor.authorFranke, Lude
dc.contributor.authorFesten, Eleonora AM
dc.contributor.authorvan der Wijst, Monique GP
dc.date.accessioned2022-06-15T15:01:01Z
dc.date.available2022-06-15T15:01:01Z
dc.date.issued2022-06-14
dc.date.submitted2021-01-19
dc.identifier.issn2045-2322
dc.identifier.others41598-022-13812-y
dc.identifier.other13812
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/338119
dc.description.abstractSingle-cell RNA sequencing (scRNA-seq) has revolutionized the study of the cellular landscape of organs. Most single-cell protocols require fresh material, which limits sample size per experiment, and consequently, introduces batch effects. This is especially true for samples acquired through complex medical procedures, such as intestinal mucosal biopsies. Moreover, the tissue dissociation procedure required for obtaining single cells is a major source of noise; different dissociation procedures applied to different compartments of the tissue induce artificial gene expression differences between cell subsets. To overcome these challenges, we have developed a one-step dissociation protocol and demonstrated its use on cryopreserved gut mucosal biopsies. Using flow cytometry and scRNA-seq analysis, we compared this one-step dissociation protocol with the current gold standard, two-step collagenase digestion, and an adaptation of a recently published alternative, three-step cold-active Bacillus licheniformus protease digestion. Both cell viability and cell type composition were comparable between the one-step and two-step collagenase dissociation, with the former being more time-efficient. The cold protease digestion resulted in equal cell viability, but better preserves the epithelial cell types. Consequently, to analyze the rarer cell types, such as glial cells, larger total biopsy cell numbers are required as input material. The multi-step protocols affected cell types spanning multiple compartments differently. In summary, we show that cryopreserved gut mucosal biopsies can be used to overcome the logistical challenges and batch effects in large scRNA-seq studies. Furthermore, we demonstrate that using cryopreserved biopsies digested using a one-step collagenase protocol enables large-scale scRNA-seq, FACS, organoid generation and intraepithelial lymphocyte expansion.
dc.languageen
dc.publisherSpringer Science and Business Media LLC
dc.subjectArticle
dc.subject/692/308/2056
dc.subject/631/337/2019
dc.subjectarticle
dc.titleGut mucosa dissociation protocols influence cell type proportions and single-cell gene expression levels.
dc.typeArticle
dc.date.updated2022-06-15T15:01:01Z
prism.issueIdentifier1
prism.publicationNameSci Rep
prism.volume12
dc.identifier.doi10.17863/CAM.85528
dcterms.dateAccepted2022-05-27
rioxxterms.versionofrecord10.1038/s41598-022-13812-y
rioxxterms.versionVoR
rioxxterms.licenseref.urihttp://creativecommons.org/licenses/by/4.0/
dc.contributor.orcidUniken Venema, Werna TC [0000-0002-1515-0920]
dc.contributor.orcidJonkers, Iris [0000-0003-2304-7939]
dc.contributor.orcidGhouraba, Mennatallah [0000-0003-4671-3500]
dc.contributor.orcidvan der Wijst, Monique GP [0000-0003-1520-3970]
dc.identifier.eissn2045-2322
cam.issuedOnline2022-06-14


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