In a previous study, seed coat and cotyledon tissues of Williams, Richland and T157 soybean lines were investigated to show tissue specificity of CHS siRNA expression (Tuteja et al., 2009). Here, we investigated more tissues such as leaf, root and germinating cotyledon to ascertain the tissue specificity of CHS siRNAs in Williams. Data from multiple small RNA libraries were sequenced deeply by the Illumina high-throughput sequencing technology. The total numbers of small RNA reads were from three million to thirty million, providing sufficient data to show the tissue specificity of CHS siRNA. Overall design: High-throughput sequencing using Genome Analyzer II and Illumina HiSeq 2000 was performed.