Constructing High Quality RNA-Seq Libraries from Limited Amounts of Total RNA

An ever-present challenge for Next Generation Sequencing (NGS) applications is obtaining high-quality data from low amounts of starting material. The large number of processing steps required to generate RNA-Seq libraries presents additional challenges for efficiency and retention of material throughout each enzymatic processing and cleanup step. Here the robustness of the NEXTflex™ Poly(A) Beads and the NEXTflex™ Rapid Directional RNA-Seq Kit for mRNA isolation and library preparation was examined using a dilution series of total RNA isolated from a human cell line. Several metrics of library quality, including unique reads, ribosomal RNA contamination and the number of transcripts detected were analyzed to determine the quality of low-input total RNA samples. The data demonstrate that high-quality libraries are readily produced from as little as 10 ng total RNA using the NEXTflex Rapid Directional RNA-Seq Kit.