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Full-length RNA-seq from single cells using Smart-seq2
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DOI：10.1038/nprot.2014.006.png)
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Abstract
En 中文
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 similar to 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.
Keywords:
REVERSE-TRANSCRIPTASE
SECONDARY STRUCTURE
GENE-EXPRESSION
RT-PCR
HETEROGENEITY
BETAINE
TUMOR
CDNA
CONSTRUCTION
LANDSCAPE
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