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Method for constructing Ribo-seq sequencing libraries

A ribo-seq and sequencing library technology, applied in chemical libraries, biochemical equipment and methods, microbial measurement/testing, etc., can solve problems such as large applicability

Active Publication Date: 2018-10-09
CHI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using this method, it is possible to directly build a library without removing rRNA, and at the same time avoid contamination of small RNA and non-translated RNA in the cell, simplify the operation process, and have great applicability. There is no need to synthesize rRNA capture reagents according to different species, which greatly reduces the cost of building Ribo-seq libraries At the same time, good quality RPF sequencing data can be obtained, which can effectively overcome the technical defects of the current method, such as high sequencing cost, complicated operation, and limited applicability to several species.

Method used

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  • Method for constructing Ribo-seq sequencing libraries
  • Method for constructing Ribo-seq sequencing libraries
  • Method for constructing Ribo-seq sequencing libraries

Examples

Experimental program
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Embodiment 1

[0087] (1) Escherichia coli BW25113 glycerol species was streaked on the LB plate and cultivated overnight;

[0088] (2) Pick a single colony on the plate, inoculate in 3ml LB, and activate overnight at 37°C and 200rpm;

[0089] (3) Inoculate the activated seed liquid with an inoculum amount of 1%, inoculate it in 50ml of LB, and cultivate it at 37°C and 200rpm until the culture medium OD 600 =0.6;

[0090] (4) Add chloramphenicol with a final concentration of 100 μg / ml to the culture medium obtained in the upward step, shake it in ice water for 5 minutes to rapidly cool to 4°C, centrifuge at 5000g for 5 minutes at 4°C, and remove the supernatant;

[0091] (5) Add pre-cooled 50ml PBS (100μg / ml chloramphenicol) to the precipitate obtained in the upward step, resuspend the precipitate, centrifuge at 5000g at 4°C for 5min to collect the bacteria, and discard the supernatant;

[0092] (6) Repeat step (5);

[0093] (7) Add pre-cooled 5.4ml lysozyme (lysozyme) buffer to the preci...

Embodiment 2

[0124] (1) Human lung cancer A549 cells (1 million) frozen at -80°C were thawed in ice water for 30 minutes;

[0125] (2) Add pre-cooled PBS to resuspend the A549 cells obtained in the previous step, and centrifuge at 4000g for 5min at 4°C;

[0126] (3) Add 2ml of lysis Buffer to the cell pellet obtained in the previous step, let stand on ice for 30min, and centrifuge at 16800rpm for 15min at 4°C;

[0127] (4) with embodiment 1 step (9);

[0128] (5) Slowly absorb the supernatant with a pipette gun, add 0.2ml of pre-cooled M buffer Mix (50mM Tris-HCl (pH=7.9), 5mM CaCl 2 , 6mM MgCl 2 , 100μg / ml cycloheximide, 1X BSA) to wash the tube wall other than the transparent precipitate, discard it, then add 0.25ml of pre-cooled M buffer Mix to gently blow and resuspend the transparent precipitate, transfer it to an EP tube, and obtain RNC heavy suspension;

[0129] Steps (6)-(10) are respectively successively with embodiment 1 steps (11)-(15);

[0130] (11) In order to check the r...

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Abstract

The invention discloses a method for constructing Ribo-seq sequencing libraries. The method includes separating Ribosome-nascent chain complexes (RNC); carrying out enzyme digestion on the RNC; directly constructing the libraries without rRNA [ribosomal RNA (ribonucleic acid)] removal; selecting RPF (ribosome protected fragments) bands and carrying out sequencing on RPF. The method has the advantages that rRNA does not need to be removed, accordingly, expensive rRNA removal reagent kits can be omitted, optional species can be coped, and the method is wide in application range; RPF reads proportions of the method are higher than RPF reads proportions of the traditional methods, the flux requirements can be lowered, and accordingly the sequencing cost can be lowered.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing a Ribo-seq sequencing library and the constructed sequencing library. Background technique [0002] Ribosome profiling (also known as Ribo-seq, "ribosome profile" or "ribosome distribution profile") technology is a method of analyzing the position of each ribosome on mRNA by next-generation sequencing. In this method, nucleases are used to degrade mRNA fragments not covered by ribosomes, and then ribosomes are removed, and ribosome-covered fragments (Ribosome protected fragments, RPF) are determined by next-generation sequencing. Since each RPF corresponds to a ribosome, after aligning these RPFs to the reference sequence, the position of each ribosome in translation can be known, so as to study translation globally. [0003] Although this technology can enable people to study translation from a global perspective, it has problems such as poor controllabili...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C40B50/06C12Q1/6869
CPCC12Q1/6806C12Q1/6869C40B50/06C12Q2535/122
Inventor 张鸿张弓赵晶金静洁
Owner CHI BIOTECH CO LTD
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