An optimized protocol to identify keratinocyte subpopulations in vitro by single-cell RNA sequencing analysis
Summary: Here, we describe a protocol for single-cell isolation from the primary culture of normal human epidermal keratinocytes derived from neonatal foreskin. The cell culture conditions have been optimized for inducing expression of keratinocyte differentiation markers. Cells are cultured in the...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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Elsevier
2022-12-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166722007869 |
| _version_ | 1798005498482524160 |
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| author | Ratklao Siriwach Anh Quynh Ngo Shuh Narumiya Dean Thumkeo |
| author_facet | Ratklao Siriwach Anh Quynh Ngo Shuh Narumiya Dean Thumkeo |
| author_sort | Ratklao Siriwach |
| collection | DOAJ |
| description | Summary: Here, we describe a protocol for single-cell isolation from the primary culture of normal human epidermal keratinocytes derived from neonatal foreskin. The cell culture conditions have been optimized for inducing expression of keratinocyte differentiation markers. Cells are cultured in the absence or presence of a bioactive lipid lysophosphatidic acid (LPA). Single cells are isolated by Fluidigm C1 system. This is followed by cDNA library preparation using Takara SMART-Seq v4 Ultra and Illumina Nextera XT kit for RNA sequencing.For complete details on the use and execution of this protocol, please refer to Siriwach et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
| first_indexed | 2024-04-11T12:41:20Z |
| format | Article |
| id | doaj.art-c207a63beeaf40e28b314f36c206ddd9 |
| institution | Directory Open Access Journal |
| issn | 2666-1667 |
| language | English |
| last_indexed | 2024-04-11T12:41:20Z |
| publishDate | 2022-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | STAR Protocols |
| spelling | doaj.art-c207a63beeaf40e28b314f36c206ddd92022-12-22T04:23:30ZengElsevierSTAR Protocols2666-16672022-12-0134101906An optimized protocol to identify keratinocyte subpopulations in vitro by single-cell RNA sequencing analysisRatklao Siriwach0Anh Quynh Ngo1Shuh Narumiya2Dean Thumkeo3Department of Drug Discovery Medicine, Kyoto University Graduate School of Medicine, Kyoto 606-8507, JapanDepartment of Drug Discovery Medicine, Kyoto University Graduate School of Medicine, Kyoto 606-8507, JapanDepartment of Drug Discovery Medicine, Kyoto University Graduate School of Medicine, Kyoto 606-8507, JapanDepartment of Drug Discovery Medicine, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan; Corresponding authorSummary: Here, we describe a protocol for single-cell isolation from the primary culture of normal human epidermal keratinocytes derived from neonatal foreskin. The cell culture conditions have been optimized for inducing expression of keratinocyte differentiation markers. Cells are cultured in the absence or presence of a bioactive lipid lysophosphatidic acid (LPA). Single cells are isolated by Fluidigm C1 system. This is followed by cDNA library preparation using Takara SMART-Seq v4 Ultra and Illumina Nextera XT kit for RNA sequencing.For complete details on the use and execution of this protocol, please refer to Siriwach et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.http://www.sciencedirect.com/science/article/pii/S2666166722007869Sequence analysisCell BiologySingle CellDevelopmental biologyRNAseq |
| spellingShingle | Ratklao Siriwach Anh Quynh Ngo Shuh Narumiya Dean Thumkeo An optimized protocol to identify keratinocyte subpopulations in vitro by single-cell RNA sequencing analysis STAR Protocols Sequence analysis Cell Biology Single Cell Developmental biology RNAseq |
| title | An optimized protocol to identify keratinocyte subpopulations in vitro by single-cell RNA sequencing analysis |
| title_full | An optimized protocol to identify keratinocyte subpopulations in vitro by single-cell RNA sequencing analysis |
| title_fullStr | An optimized protocol to identify keratinocyte subpopulations in vitro by single-cell RNA sequencing analysis |
| title_full_unstemmed | An optimized protocol to identify keratinocyte subpopulations in vitro by single-cell RNA sequencing analysis |
| title_short | An optimized protocol to identify keratinocyte subpopulations in vitro by single-cell RNA sequencing analysis |
| title_sort | optimized protocol to identify keratinocyte subpopulations in vitro by single cell rna sequencing analysis |
| topic | Sequence analysis Cell Biology Single Cell Developmental biology RNAseq |
| url | http://www.sciencedirect.com/science/article/pii/S2666166722007869 |
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