Ribosome print sequencing library construction method

A technology of sequencing library and construction method, applied in the field of ribosome imprinted sequencing library construction, can solve the problems of restricting the development and promotion of ribosome imprinted sequencing, and achieve the effects of reducing material cost, keeping the theoretical level consistent, and improving stability

Pending Publication Date: 2021-06-04
JINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These factors have greatly limited the development and promotion of ribosomal imprinted sequencing technology. Therefore, the current ribosomal imprinted sequencing technology urgently needs a library construction solution that can be standardized, easy to operate and has high robustness.

Method used

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  • Ribosome print sequencing library construction method
  • Ribosome print sequencing library construction method
  • Ribosome print sequencing library construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] A fast, robust and high-precision ribosome imprinted sequencing library construction method, the specific steps are as follows:

[0041] 1. Obtain the RFP-RNA of Saccharomyces cerevisiae S288C strain (see the literature for the experimental steps: Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS (2012) The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments . Nat Protoc 7(8):1534–1550.). The obtained 20ng RFP-RNA was subjected to RFP-RNA double-end repair reaction.

[0042]2. Use T4 PNK polynucleotide kinase to dephosphorylate the 3' end, and react at 37°C for 60min. The reaction system is shown in Table 1.

[0043] Table 1

[0044]

[0045] 3. Carry out the next reaction in the reaction solution after dephosphorylation treatment, use T4 PNK polynucleotide kinase to perform 5' terminal phosphorylation treatment, and react at 37°C for 60min. The reaction system is shown in Table 2.

[0046...

Embodiment 2

[0062] A fast, robust and high-precision ribosome profiling sequencing library construction method, the specific steps are as follows:

[0063] 1. Obtain the RFP-RNA of Pichia pastoris GS115 strain (see the literature for the experimental steps: Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS (2012) The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc 7(8):1534–1550.). The obtained 20ng RFP-RNA was subjected to RFP-RNA double-end repair reaction.

[0064] 2. Use T4 PNK polynucleotide kinase to dephosphorylate the 3' end, and react at 37°C for 60min. The reaction system is shown in Table 3.

[0065] table 3

[0066]

[0067] 3. Carry out the next reaction in the reaction solution after dephosphorylation treatment, use T4 PNK polynucleotide kinase to perform 5' terminal phosphorylation treatment, and react at 37°C for 60min. The reaction system is shown in Table 4.

[0068] Table 4...

Embodiment 3

[0084] A fast, robust and high-precision ribosome profiling sequencing library construction method, the specific steps are as follows:

[0085] 1. Obtain the RFP-RNA of the human lung adenocarcinoma cell line A549 (see the literature for the experimental steps: Jang, C., Lahens, N.F., Hogenesch, J.B. & Sehgal, A. Ribosome profiling reveals an important role for translational control in circadian gene expression. Genome Res. 25, 1836–1847(2015).). The obtained 20ng RFP-RNA was subjected to RFP-RNA double-end repair reaction.

[0086] 2. Use T4 PNK polynucleotide kinase to dephosphorylate the 3' end, and react at 37°C for 60min. The reaction system is shown in Table 5.

[0087] table 5

[0088]

[0089] 3. Carry out the next reaction in the reaction solution after dephosphorylation treatment, use T4 PNK polynucleotide kinase to perform 5' terminal phosphorylation treatment, and react at 37°C for 60min. The reaction system is shown in Table 6.

[0090] Table 6

[0091] ...

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Abstract

The invention provides a ribosome print sequencing library construction method. The library construction method provided by the invention comprises the following steps: (1) extracting RFP-RNA from a sample; (2) repairing structures of the two ends of the RFP-RNA; and (3) subjecting normal-structure RNA to library construction by employing a small RNA library construction kit. According to the method, the RFP-RNA can restore to a normal RNA structure through repairing the two ends of the RFP-RNA, the normal RNA structure can be in seamless engagement with the small RNA kit on the market for direct library construction, rRNA is not required to be removed, a complicated RFP-RNA library construction method is not required to be employed, experimental steps for library construction are simplified, the material cost is reduced, and the experimental stability is improved; and the experimental time is shortened by 4-5 days, and the time cost is reduced by 50%. The library construction method provided by the invention is applicable to all classes of species; and obtained sequencing data are high in quality and are consistent with a theoretical level.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a ribosome imprinted sequencing library. Background technique [0002] Ribosome profiling, Ribo-seq, RFP, RPF technology was developed by Ingolia et al. 324, 218–223 (2009).) First published in 2009. This technology uses nuclease to digest the non-ribosome-protected RNA in the lysate of biological samples. After ultracentrifugation and purification, single ribosomes and their protected RNA fragments are obtained. Trizol is used to extract RNA, and then urea-acrylamide gel is used The 18nt-35nt RFP-RNA was purified by electrophoresis, and finally the sequencing library was constructed by next-generation sequencing, and the ribosome-protected RNA fragment (18-35nt) was sequenced to obtain all ribosomes in the organism under certain physiological conditions. specific location information on . High-precision ribosome profiling data can be used for systematic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6869C40B50/06
CPCC12Q1/6806C40B50/06C12Q1/6869
Inventor 卢小龙张弓陈洋金静洁
Owner JINAN UNIVERSITY
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