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, we 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. We 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.