Gene-expression profiling by Smart-3SEQ v1

This protocol combines aspects of 3SEQ (Beck et al., PLOS ONE 2010), Smart-seq2 (Picelli et al. Nat Methods 2013), and UMIs (Kivioja et al., Nat Methods 2011) into a streamlined method for making a sequencing library to measure the expression of polyadenylated RNAs. It is fast (3 hr), inexpensive (< $10 / library), sensitive (tested successfully with 10 pg human total RNA, equivalent to one cell), and scalable (all steps performed in PCR tubes or microplates). It generates digital gene-expression data equivalent to SEQ's: one read per RNA molecule in the starting material, at the 3' end of the gene, which allows simpler quantification of transcript abundance at the expense of most information about splicing isoforms.