title: Highly multiplexed and strand-specific single-cell RNA 5' end sequencing.
abstract: Single-cell analysis of gene expression is increasingly important for the analysis of complex tissues, including cancer, developing organs and adult stem cell niches. Here we present a detailed protocol for quantitative gene expression analysis in single cells, by the sequencing of mRNA 5' ends. In all, 96 cells are lysed, and their mRNA is converted to cDNA. By using a template-switching mechanism, a bar code and an upstream primer-binding sequence are introduced simultaneously with reverse transcription. All cDNA is pooled and then prepared for 5' end sequencing, including fragmentation, adapter ligation and PCR amplification. The chief advantage of this approach is the great reduction in cost and time, afforded by the early bar-coding strategy. Compared with previous methods, it is more suitable for large-scale quantitative analysis, as well as for the characterization of transcription start sites, but it is unsuitable for the detection of alternatively spliced transcripts. Sample preparation takes 3 d, and two sets of 96 cells can be prepared in parallel. Finally, the sequencing and data analysis can take an additional 4 d altogether.
authors.affiliation: Laboratory for Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Saiful
authors.initials: S
authors.lastname: Islam
authors.forename: Una
authors.initials: U
authors.lastname: Kjällquist
authors.forename: Annalena
authors.initials: A
authors.lastname: Moliner
authors.forename: Pawel
authors.initials: P
authors.lastname: Zajac
authors.forename: Jian-Bing
authors.initials: JB
authors.lastname: Fan
authors.forename: Peter
authors.initials: P
authors.lastname: Lönnerberg
authors.forename: Sten
authors.initials: S
authors.lastname: Linnarsson
mesh_terms: D020121:5' Untranslated Regions; D018076:DNA, Complementary / Q000737:chemistry; D005434:Flow Cytometry; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000737:chemistry*; D017423:Sequence Analysis, RNA / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*
chemical_list: D020121:5' Untranslated Regions; D018076:DNA, Complementary; D012333:RNA, Messenger