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

BACKGROUND
Deep 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.


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
We 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.


Introduction
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 have previously reported a high-throughput RNA sequencing strategy that has significantly lowered the library preparation cost [2]. Here we sought to develop an alternative protocol that would also 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. The 5' adapter is a conventional RNA oligo that can be synthesized by commercial oligo manufacturers. The 3' adapter is a modified DNA oligo containing a 5'5'-adenyl pyrophosphoryl moiety and a 3' blocking group such as amine or dideoxylnucleotides. The adenyl modification on 3' adapter is crucial for the library preparation as it enables the adapter to be ligated to RNA in the absence of ATP by a truncated form of T4 RNA ligase 2. This effectively prevents small RNA self-ligation and concatenation. Despite the simplicity of this method, the adenylated DNA oligos are very costly to synthesize, and few are available commercially.
It has been reported that T4 DNA ligase can convert DNA oligos to pre-adenylated adapters [4]. In a ligation reaction, T4 DNA ligase first adenylates the donor DNA that has a 5' phosphate, and the adenylated intermediate reacts with the acceptor DNA with a free 3' hydroxyl group, resulting in phosphodiester bond formation. This process could be interrupted, allowing the adenylated intermediate to be purified. However, the adenylation efficiency of T4 DNA ligase is inconsistent and several improvements have been suggested to optimize the reaction [5]. We reason that the difficulties of utilizing T4 DNA ligase to generate adenylated adapters are partly due to the fact that it works best on doublestranded substrate, while the desired adapter is a singlestranded molecule. Hence, an RNA ligase could be a more suitable enzyme for adapter adenylation, since RNA ligation also generates a 5' adenylated donor intermediate [6].
In this report, we demonstrate that T4 RNA ligase 1 can efficiently adenylate DNA oligos with 5' phosphate and 3' blocking group, and we have optimized the reaction condition for large-scale adapter production. Using the enzymatically adenylated adapters, we developed a rapid and cost-effective protocol to generate small RNA sequencing libraries. 3' multiplex adapter: Phosphate-CAGATCGGAAGAGC ACACGT-amine.
Multiplex reverse PCR primers with 6 nt barcode (NNNNNN indicates the location of TruSeq index sequences): CAAGCAGAAGACGGCATACGAGAT-(NNNNNN)-G TGACTGGAGTTCAGACGTGTGCTCTTCCGATC* T.   has a 5' phosphate. Successfully ligated small RNA could be visualized on Urea-PAGE ( Figure 1A). 8. Add 0.5 uL reverse transcription primer (20 uM) to the ligation reaction, heat inactivate the reaction at 65°C for 15 min and incubate the reaction at 25°C for 10 min. 9. Optional: to minimize adapter dimer formation in the subsequent PCR enrichment, the ligated sRNA could be PAGE purify before proceeding to the following 5' adapter ligation steps.

5' adapter ligation
10. Denature the 5' adapter (10 uM, dissolved in RNase-free water) at 65°C for 30 sec, immediately chill on ice. 11. Setup the 5' ligation reaction with 20.5 uL 3' ligation product, 1 uL 5' adapter (10 uM), 1 uL 10x T4 RNA ligase 1 buffer with ATP, 0.5 uL RNase Inhibitor, 2 uL 10 mM ATP, 4 uL RNase-free water and 1 uL T4 RNA ligase 1. The reaction is incubated at 25°C for 4 h or 18°C overnight. Note: the 3' end of the small RNA has already been ligated to the 3' adapter that has an amine group at the 3' end, and could no longer take part in the ligation reaction; thus its 5' end could be ligated to an RNA oligo in the presence of ATP.
13. Incubate the reaction at 50°C for 1 h, and heat inactivate the reverse transcriptase at 75°C for 15 min.

PCR enrichment
14. Assemble the PCR reaction with 10 uL RT product, 1 uL forward primer, 1 uL reverse primer, 1 uL dNTP, 6 uL 5x Phusion HF buffer, 10.5 uL water and 0.5 uL Phusion DNA polymerase. The remaining RT product could be stored at −20°C. 15. Perform 12 to 15 cycles of PCR amplification as followed: 94°C 2 min, 12-15 cycles of 98°C for 12 sec, 65°C for 30 sec and 72°C for 30 sec, and a final extension at 72°C for 2 min. 16. Seperate the PCR product on a 2% agarose gel.
Note: The multiplex sRNA-Seq library and adapter dimer are 150 and 120 bp respectively ( Figure 1B); whereas the reverse transcription and PCR primers used in the Illumina non-multiplex library preparation lack the barcode sequences, hence the sizes of non-multiplex sRNA-Seq library and adapter dimer are smaller at 120 and 90 bp, respectively. 17. Cut the band corresponding to the small RNA library and gel-purify the product with Qiagen MinElute kit. 18. Optional step: adjust PCR cycles accordingly to avoid over or insufficient PCR amplification.

Quantify and pool libraries for sequencing
19. Quantify the purified library using QuaniT HS DNA assay kit.

Comments
It has been shown that T4 RNA ligase 1 could be used to adenylated RNA oligos [7,8]. To examine its adenylation efficiency on DNA substrates, we have synthesized a 5' phosphorylated and 3' amine blocked single-stranded DNA substrate (p-CTGTAGGCACCAT CAAT-amine) according to the first published micro-RNA cloning adapter [3]. Since its 3' end is blocked by the amine modification, when its 5' end is activated by adenylation, there is no free 3' hydroxyl group available for ligation, leading to accumulation of the adenylated intermediate. We incubated the substrate with T4 RNA ligase 1 under various reaction conditions, and in all cases, a clear band shift was observed on the denatured PAGE indicating successful adenylation ( Figure 2). We found that adenylation reactions at room temperature with 10 μM of substrate and in the presence of 20% PEG gave the best result. It has been shown that T4 RNA ligase 1 adenylates ribonucleotides with different efficiencies, and rCTP is its best substrate [6][7][8]. We examined its ability to act on DNA substrates with different 5' deoxylnucleotides, and found that oligos starting with a 5' dC were efficiently adenylated ( Figure 2B). Hence we have designed all 3' adapters with a 5' dC to maximize the adenylation efficiency. Unlike the T4 DNA ligase-based method that requires time-consuming PAGE separation of the adenylated product from other oligos used in the reaction, oligos adenylated by T4 RNA ligase 1 can be directly purified using a Sephadex buffer exchange column or commercial kits. We have generated a sRNA-Seq library for tomato fruit using the T4 RNA ligase 1 adenylated adapter, and the result matches the published tomato small RNA datasets [9] generated by Illumina reagents (Figure 3). This protocol has also been adopted for small RNA sequencing in different plant species including sweet potato, pepper, watermelon, and cowpea.