Directional RNA-seq libraries from small samples without pre-amplification for the quantification of lncRNAs

Development of high-throughput sequencing technologies has uncovered the immensity of the long noncoding RNA (lncRNA) world. Divergently transcribed lncRNAs from bidirectional gene promoters, called promoter-associated noncoding RNAs (pancRNAs), account for ~20% of the total number of lncRNAs, and this major fraction is involved in many biological processes, such as development and cancer formation.

Recently, researchers at Kyushu University have found that the pancRNAs activate their partner genes, as represented by the fact that pancIl17d, a pancRNA that is transcribed from the antisense strand of the promoter region of Interleukin 17d (Il17d) at the onset of zygotic gene activation (ZGA), is essential for mouse preimplantation development through Il17d upregulation. The discovery of the expression of a specific set of pancRNAs during ZGA was achieved by using a method that generates directional RNA-seq libraries from small-scale samples. Although there are several methods available for small-scale samples, most of them require a pre-amplification procedure that frequently generates some amplification biases toward a subset of transcripts. The researchers provide here a highly sensitive and reproducible method based on the preparation of directional RNA-seq libraries from as little as 100 mouse oocytes or embryos without pre-amplification for the quantification of lncRNAs as well as mRNAs.

A schematic view of the directional RNA-seq from small-scale samples

Oocytes or embryos are directly put into lysis and polyA extraction buffer. polyA+ RNAs extracted by magnetic oligo dT beads are eluted and fragmented. These fragmented RNAs are subjected to reverse transcription with random primers to generate first-strand cDNAs. Using these single-strand cDNAs as templates, dUTP-containing second-strand cDNAs are synthesized. Circulizing adaptors are ligated by TA ligation reaction. In the reaction of USER enzyme, this circularized adaptor becomes linear and the dUTP-containing second-strand becomes fragmented. As a result, only first-strand cDNAs are amplified by the subsequent PCR reaction. During this PCR reaction, sequencing tags are added. These constructed libraries can immediately be subjected to high-throughput sequencing