Genome-wide study of gene expression, or transcriptome profiling, is critical for our understanding of biological functions, including developmental processes. Recent breakthroughs in high-throughput sequencing technologies have revolutionized gene expression profiling to study the transcriptome at the nucleotide level, which is known as RNA-seq. RNA-seq, also called “whole transcriptome shotgun sequencing,” uses next-generation sequencing technologies to sequence cDNA in order to infer a sample’s RNA content. Here we describe a detailed bench-ready protocol to generate RNA-seq libraries for high-throughput single-end or pair-end sequencing compatible with the Illumina sequencing platform.