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Nat Protoc. 2014 Jan;9(1):171-81. doi: 10.1038/nprot.2014.006. Epub 2014 Jan 2.

Full-length RNA-seq from single cells using Smart-seq2.

Author information

1
Ludwig Institute for Cancer Research, Stockholm, Sweden.
2
1] Ludwig Institute for Cancer Research, Stockholm, Sweden. [2] Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.

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.

PMID:
24385147
DOI:
10.1038/nprot.2014.006
[Indexed for MEDLINE]

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