A cost-effective method for Illumina small RNA-Seq library preparation using T4 RNA ligase 1 adenylated adapters

Yun-Ru Chen,Zhangjun Fei,Yi Zheng,Bao Liu,Silin Zhong,Jim Giovannoni

doi:10.1186/1746-4811-8-41

Yun-Ru Chen, Zhangjun Fei + Show 4 more

Open Access

https://doi.org/10.1186/1746-4811-8-41

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Abstract

BackgroundDeep sequencing is a powerful tool for novel small RNA discovery. Illumina small RNA sequencing library preparation requires a pre-adenylated 3’ end adapter containing a 5’,5’-adenyl pyrophosphoryl moiety. In the absence of ATP, this adapter can be ligated to the 3’ hydroxyl group of small RNA, while RNA self-ligation and concatenation are repressed. Pre-adenylated adapters are one of the most essential and costly components required for library preparation, and few are commercially available.ResultsWe demonstrate that DNA oligo with 5’ phosphate and 3’ amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum) using the T4 RNA ligase 1 adenylated adapter.ConclusionWe provide an efficient and low-cost method for small RNA sequencing library preparation, which takes two days to complete and costs around $20 per library. 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.

Highlights

The rapidly increasing NextGen sequencing capacity is enabling researchers to combine more samples for multiplexed sequencing, and the sequencing cost itself could be less than that of the library preparation [1]
We sought to develop an alternative protocol that would simplify the small RNA library preparation. We note that both the Illumina small RNA sequencing and directional mRNA sequencing library construction protocols are based on the early microRNA cloning strategies [3], in which two adapters are sequentially attached to the RNA molecule to create the priming sites for subsequent PCR amplification
It has been reported that T4 DNA ligase can convert DNA oligos to pre-adenylated adapters [4]

Summary

Results

We demonstrate that DNA oligo with 5’ phosphate and 3’ amine groups can be enzymatically adenylated by T4 RNA ligase 1 to generate customized pre-adenylated adapters. We have constructed and sequenced a small RNA library for tomato (Solanum lycopersicum) using the T4 RNA ligase 1 adenylated adapter

Conclusion

Introduction

Materials and methods

15. Perform 12 to 15 cycles of PCR amplification as followed