Directly Ligate Sequencing Adapters to the First-Strand cDNA

DLAF is a strand-specific RNA sequencing method that avoids second-strand cDNA synthesis by directly attaching unique double-stranded adapters to the first-strand cDNA (Agarwal et al., 2015). First, mRNA is isolated and depleted of rRNA. Next, RNA fragments are partially hydrolyzed and annealed to random primers before RT. The resulting first-strand cDNA is purified and flanked with double-stranded adapters. One strand of each adapter, the annealing strand, contains uracils and an overhang with 5 or 6 random nucleotides that anneal to the cDNA. The other strand, the ligating strand, directly ligates to the last nucleotide of the cDNA strand. 3′-hexanediol is attached to the 3′ ends of each adapter to reduce concatenation with other strands. The adapter-ligated cDNAs are purified and exposed to uracil-specific excision enzyme, to remove the annealing strands from the cDNA. This process results in single-stranded cDNA strands flanked by the ligated adapters. The cDNA is PCR-amplified, purified, and sequenced.


  • Reads mRNA sequences solely from the first strand of cDNA
  • Sequences the 5′ and 3′ ends of mRNAs
  • Fewer steps ensure a high yield of cDNA library product


  • Unable to differentiate between 5′ capped and uncapped mRNA


Illumina Library prep and Array Kit Selector


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Iwase S., Brookes E., Agarwal S., et al. A Mouse Model of X-linked Intellectual Disability Associated with Impaired Removal of Histone Methylation. Cell Rep. 2016;