![]() Cut the band corresponding to the small RNA library and gel-purify the product with Qiagen MinElute kit.ġ8. 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.ġ7. Seperate the PCR product on a 2% agarose gel. Perform 12 to 15 cycles of PCR amplification as followed: 94☌ 2 min, 12–15 cycles of 98☌ for 12 sec, 65☌ for 30 sec and 72☌ for 30 sec, and a final extension at 72☌ for 2 min.ġ6. The remaining RT product could be stored at −20☌.ġ5. 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. Using the enzymatically adenylated adapters, we developed a rapid and cost-effective protocol to generate small RNA sequencing libraries.ġ4. 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. Hence, an RNA ligase could be a more suitable enzyme for adapter adenylation, since RNA ligation also generates a 5’ adenylated donor intermediate. 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 double-stranded substrate, while the desired adapter is a single-stranded molecule. ![]() However, the adenylation efficiency of T4 DNA ligase is inconsistent and several improvements have been suggested to optimize the reaction. This process could be interrupted, allowing the adenylated intermediate to be purified. 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. It has been reported that T4 DNA ligase can convert DNA oligos to pre-adenylated adapters. Despite the simplicity of this method, the adenylated DNA oligos are very costly to synthesize, and few are available commercially. This effectively prevents small RNA self-ligation and concatenation. 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. 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 5’ adapter is a conventional RNA oligo that can be synthesized by commercial oligo manufacturers. We note that both the Illumina small RNA sequencing and directional mRNA sequencing library construction protocols are based on the early microRNA cloning strategies, in which two adapters are sequentially attached to the RNA molecule to create the priming sites for subsequent PCR amplification. ![]() Here we sought to develop an alternative protocol that would also simplify the small RNA library preparation. We have previously reported a high-throughput RNA sequencing strategy that has significantly lowered the library preparation cost. 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.
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