A team of researchers at USDA and Cornell University provide an efficient and low-cost method for small RNA sequencing library preparation for plants, which takes two days to complete and costs around $20 per library.
Current small RNA sequencing and directional mRNA sequencing library construction protocols require two adapters to be sequentially attached to the RNA molecule to create the priming sites for subsequent PCR amplification. These adapters are modified, adenylated DNA oligos which are very costly to synthesize. We demonstrate that DNA oligos with 5′ phosphate and 3′ amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters, thus eliminating expensive synthesis requirements.
This protocol has been tested in several plant species for small RNA sequencing including sweet potato, pepper, watermelon, and cowpea, and could be readily applied to any RNA samples.
- Chen YR, Zheng Y, Liu B, Zhong S, Giovannoni J, Fei Z. (2012) A cost-effective method for Illumina small RNA-Seq library preparation using T4 RNA ligase 1 adenylated adapters. Plant Methods 8(1), 41. [article]