Nat Commun. 2018 Feb 12;9(1):619. doi:10.1038/s41467-018-02866-0

Hayashi T, Ozaki H, Sasagawa Y, Umeda M, Danno H, Nikaido I.

Single-cell full-length total RNA sequencing uncovers dynamics of recursive splicing and enhancer RNAs.

RamDA-seq. First-strand cDNAs are synthesised with a RNAse H minus reverse-transcriptase. DNAse I introduces nicks that prime synthesis of new cDNA molecules. T4gp32 promotes the strand-displacement activity of the reverse-transcriptase. Second-strand cDNAs are synthethised with Klenow fragment (3′ → 5′ exo-) primed with NSRs. In single cells, genomic DNA needs to be removed because of the DNAseI digestion during RT and the low-complexity priming of the second-strand synthesis with NSRs. The resulting DNA molecules are tagmented and sequenced with standard methods. Thus, the method is non-stranded. Despite the use of NSRs, the rRNA rate stays between 20 and 30 %.