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

S Picelli, OR Faridani, ÅK Björklund, G Winberg… - Nature protocols, 2014 - nature.com
Nature protocols, 2014nature.com
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 …
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.
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