title: Full-length RNA-seq from single cells using Smart-seq2.
abstract: Emerging methods for the accurate quantification of gene expression in individual cells hold promise for revealing the extent, function and origins of cell-to-cell variability. Different high-throughput methods for single-cell RNA-seq have been introduced that vary in coverage, sensitivity and multiplexing ability. We recently introduced Smart-seq for transcriptome analysis from single cells, and we subsequently optimized the method for improved sensitivity, accuracy and full-length coverage across transcripts. Here we present a detailed protocol for Smart-seq2 that allows the generation of full-length cDNA and sequencing libraries by using standard reagents. The entire protocol takes ∼2 d from cell picking to having a final library ready for sequencing; sequencing will require an additional 1-3 d depending on the strategy and sequencer. The current limitations are the lack of strand specificity and the inability to detect nonpolyadenylated (polyA(-)) RNA.
authors.affiliation: Ludwig Institute for Cancer Research, Stockholm, Sweden.
authors.forename: Simone
authors.initials: S
authors.lastname: Picelli
authors.affiliation: Ludwig Institute for Cancer Research, Stockholm, Sweden.
authors.forename: Omid R
authors.initials: OR
authors.lastname: Faridani
authors.affiliation: 1] Ludwig Institute for Cancer Research, Stockholm, Sweden. [2] Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Asa K
authors.initials: AK
authors.lastname: Björklund
authors.affiliation: 1] Ludwig Institute for Cancer Research, Stockholm, Sweden. [2] Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Gösta
authors.initials: G
authors.lastname: Winberg
authors.affiliation: 1] Ludwig Institute for Cancer Research, Stockholm, Sweden. [2] Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Sven
authors.initials: S
authors.lastname: Sagasser
authors.affiliation: 1] Ludwig Institute for Cancer Research, Stockholm, Sweden. [2] Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Rickard
authors.initials: R
authors.lastname: Sandberg
mesh_terms: D015723:Gene Library; D017423:Sequence Analysis, RNA / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods*