Method for constructing prokaryote RNA sequencing library

A prokaryotic and sequencing library technology, applied in the field of prokaryotic RNA sequencing library construction, can solve the problems of complex rRNA blocking primer operation, low primer retrieval efficiency and specificity, and low number of retrieved genes, and achieves low preference. , to achieve the effect of preference and avoid degradation

Pending Publication Date: 2022-06-24
BEIJING STOMATOLOGY HOSPITAL CAPITAL MEDICAL UNIV
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Problems solved by technology

This method has two disadvantages. One is that the retrieval efficiency and specificity of the primers used for reverse transcription are not high, and the number of genes retrieved will be low in low-input samples.
Second, the operation of rRNA blocking primers is complicated, and RNA samples are easily degraded, and severe RNA degradation is likely to occur before the reverse transcription step, so the success rate of library construction by this method will be lower than that of traditional methods

Method used

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  • Method for constructing prokaryote RNA sequencing library
  • Method for constructing prokaryote RNA sequencing library
  • Method for constructing prokaryote RNA sequencing library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Construction of cultured Escherichia coli RNA sequencing library

[0049] 1. Total RNA extraction.

[0050] Bacteria were collected by centrifugation. Bacterial total RNA was extracted using Tiangen culture cell / bacterial total RNA extraction kit (DP430).

[0051] 2. Connect a specific base sequence at the 3' end of bacterial RNA (that is, the extended sequence of this embodiment, such as the nucleotide sequence shown in SEQ ID No. 3)

[0052] 2.1 The ligation reaction solution contains: 1×T4 RNA Ligase Buffer, 0.5 μM 3'-end RNA extension sequence (SEQID No. 3: 5'- / 5rApp / NNNNNNNNNNNNNNNNCTGTCTCTTATACACATCTGACGCTGCCGA / 3ddC / -3', 5rApp represents 5'-phosphorylated ribose Nucleotide base A, 3ddC denotes 3'-end blocked deoxyribonucleotide base C, N denotes random base), 15% PEG8000, 1 U / μL RNase inhibitor, 10U / μL T4 RNA Ligase.

[0053] 2.2 Incubate at 4°C for 16 hours. Purify RNA using an RNA purification kit.

[0054] 3. Reverse transcription and cDNA amplif...

Embodiment 2

[0076] Example 2 Sequencing and data analysis of the files obtained in Example 1

[0077] 1. Perform PE100 paired-end sequencing using the Illumina NextSeq platform. Among them, the first 16 bits of the sequencing data read by read1 are molecular tags, and the 16-100 bits are the reverse complementary sequence of the original mRNA; the sequencing data read by read2 is the forward sequence of the original mRNA.

[0078] 2. After reverse complementing the 16-100 digit data of read1, compare the reference genome with the data of read2 at the same time, and count the sequencing results of each gene aligned in the reference genome. The comparison of molecular tags and their corresponding sequencing results was counted, and all the sequencing results with the same molecular tag and compared to the same gene were combined into one expression count. Summarize the expression times of all genes to generate sequencing results.

[0079] 3. Summary of results.

[0080] 3.1 The ratio of ...

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Abstract

The invention provides a method for constructing a prokaryote RNA sequencing library. According to the method, an extension sequence can be added to prokaryote mRNA, and prokaryote RNA can be efficiently called and reversely transcribed by using the extension sequence; meanwhile, according to the method, a molecular tag can be marked for each prokaryote original mRNA molecule, so that the influence of PCR preference on the detection accuracy is avoided, and a prokaryote mRNA sequencing library with low preference and high accuracy is obtained. Compared with a traditional prokaryote RNA sequencing library construction method, the method has the advantages that the constructed library is low in preference, meanwhile, the method is easy and convenient to operate, the success rate is high, and the method is also suitable for detection of samples with extremely low initial quantity (smaller than 2ng total RNA).

Description

technical field [0001] The invention relates to a method for constructing a prokaryotic RNA sequencing library, which belongs to the technical field of gene sequencing. Background technique [0002] With the improvement of gene sequencing technology and the successive development of major projects such as the Human Genome Project, the Cancer Genome Project, and the Meta-Hit Project, genome research and RNA sequencing have increasingly become powerful tools for biological and medical research. Numerous new findings in biological and medical research are discovered or validated by RNA sequencing. However, most of the current RNA research objects are eukaryotes, which is also related to the accuracy and stability of the eukaryotic RNA sequencing library construction method is better than the prokaryotic sequencing library construction method. Therefore, the prokaryotic RNA research Better support for RNA sequencing library construction methods is needed. [0003] The messenge...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/6806C40B40/06C12N15/11C12Q1/6869
CPCC40B50/06C12Q1/6806C40B40/06C12Q1/6869C12Q2531/113C12Q2563/143C12Q2563/149
Inventor 孙静华李小林
Owner BEIJING STOMATOLOGY HOSPITAL CAPITAL MEDICAL UNIV
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