Preparation of an Illumina sequencing library for gene expression analysis (mRNA-Seq) requires microgram amounts of starting total RNA or PCR-based amplification. Here we describe a protocol based on T7 linear RNA amplification that does not introduce significant bias, requires only 10 ng total RNA, and generates a directional, fully representative, whole-transcript mRNA-Seq Illumina library that is highly consistent across over three orders of magnitude of input RNA.