Systematic evaluation of RNA-Seq preparation protocol performance (RNASeq: SMARTer)

In this study, we present a comprehensive evaluation of four RNA-Seq library preparation methods. We used three standard input protocols, the Illumina TruSeq Stranded Total RNA and TruSeq Stranded mRNA kits, and a modified NuGEN Ovation v2 kit; and an ultra-low-input RNA protocol, the TaKaRa SMARTer Ultra Low RNA Kit v3. Our evaluation of these kits included quality control measures such as overall reproducibility, 5' and 3' end-bias, and the identification of DEGs, lncRNAs, and alternatively spliced transcripts. Overall, we found that the two Illumina kits were most similar in terms of recovering DEGs, and the Illumina, modified NuGEN, and TaKaRa kits allowed identification of a similar set of DEGs. However, we also discovered that the Illumina, NuGEN and TaKaRa kits each enriched for different sets of genes. Overall design: Two mESC cell lines (biological replicates) from Zbtb24 knockout (1lox/1lox) clones are compared with two wild-type (2lox/+) clones (biological replicates) using the TaKaRa SMARTer Ultra Low RNA protocol directly on cells with no RNA preparation step. Total RNA from 100 mESCs cells and 1000 mESCs cells or approximately 1 and 10 ng RNA were used respectively.