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A simple and reverse transcription technology, applied in the field of simple RNA library construction, can solve the problems of increasing the difficulty and time-consuming of RNA library construction, and achieve the effects of low cost, simple operation and small RNA loss.
Pending Publication Date: 2022-04-29
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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It takes nearly 2 hours to remove rRNA by conventional RNaseH cutting method and hybrid capture method, which greatly increases the difficulty and time-consuming of RNA library construction
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Embodiment 1
[0032] Example 1: Effect of Random Primer Length on Reverse Transcription Efficiency
specific Embodiment approach
[0033] In this example, we verified the effect of 6-20nt random primer length (No. 1-8 in Table 1) on reverse transcription efficiency. The specific implementation is as follows:
[0034] Table 2
[0035]
[0036] 94°C for 2min, 94°C-25°C for 0.1°C / s, and store at 4°C.
[0037] table 3
[0038] components Dosage The above reaction system 16μL 0.1M DTT 2μL SuperScript TM IIIRT (200U / μL, ThermoFisher)
1μL SUPERase·In TM RNase inhibitor (20U / μL)
1μL Total 20 μL
[0039] 10min at 25°C, 15min at 42°C, 5min at 85°C, and store at 4°C. Actin and 28S RNA were quantitatively analyzed by qPCR, and the quantitative primers are listed in Table 1.
[0040] The result is as figure 1 As shown, random primers with 8-14 bases have better reverse transcription efficiency.
Embodiment 2
[0041] Example 2: Establishment of a simple RNA library construction process.
[0042] In this example, we established a simple RNA library construction process. The specific implementation is as follows:
[0043] P5 joint annealing: Dissolve 100μM P5-1 and 100μM P5-2 in 1×Annealing buffer (10mM Tris-HCl, 50mM NaCl, 1mM EDTA, pH 7.9), pipette equal volume and mix, 94℃ for 5min, 94-15℃ 0.1°C / min, store at 15°C. After annealing, the linker was diluted to a final concentration of 10 μM.
[0044] 1) RNA fragmentation and ribosomal RNA removal:
[0048] 94°C for 10min, 75°C for 1min, 55°C for 1min, and store at 4°C.
[0049] 2) RNA reverse tran...
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Abstract
The invention provides a simple RNA library building method which is characterized by comprising the following steps: (1) extracting RNA in a sample, adding a reverse transcription primer and an rRNA reverse transcription hindering probe, and fragmenting; (2) carrying out RNA reverse transcription to obtain DNA/RNA hybrid double strands; (3) linker connection: connecting a blunt-end double-chain DNA linker with adenylation modification to the 3'end of the DNA/RNA hybrid chain cDNA by using a T4 DNA ligase mutant K159L; and (4) library amplification and recovery. According to the simple RNA library building method, the principle that DNA ligase mutants K159L can be efficiently connected with DNA/RNA hybrid chains is utilized, only five steps of RNA fragmentation, reverse transcription, linker connection, library amplification and magnetic bead recovery are needed, the RNA library building process is greatly simplified, and consumed time is greatly shortened. In combination with a technology for rapidly removing rRNA by using an rRNA reverse transcription hindering probe, rRNA removal and RNA library establishment can be completed in one tube. The whole process only needs 2 hours, the operation is simple, the RNA loss is small, the cost is low, and the method is very suitable for automatic RNA library building and low-abundance RNA library building.
Description
technical field [0001] The patent of the invention relates to a simple RNA library construction method, which belongs to the field of biotechnology. Background technique [0002] RNA Next-generation sequencing (RNA-seq) is a high-throughput large-scale RNA parallel sequencing technology, which can simultaneously sequence hundreds of thousands or even millions of RNA molecules for unknown Pathogen identification, biological genetic evolution analysis, gene expression differential analysis, RNA synthesis and processing analysis, etc. Therefore, RNA-seq is widely used in fields such as scientific research and disease diagnosis, and has achieved many breakthrough results. [0003] RNA NGS library construction refers to the process of converting RNA into double-stranded DNA that can be recognized by next-generation sequencers through processes such as reverse transcription and adapter ligation, and is a key step in RNA-seq. The traditional RNA library construction method is cum...
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