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High throughput library construction method for small RNA of prokaryote

A construction method and prokaryotic technology are applied in the field of high-throughput sequencing library construction of prokaryotic small RNAs, which can solve the problem of no kits, and achieve the effect of reducing purification operations and reducing the probability of RNA degradation.

Inactive Publication Date: 2016-02-03
BIOMARKER TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there is no mature kit for prokaryotic small RNA sequencing

Method used

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  • High throughput library construction method for small RNA of prokaryote
  • High throughput library construction method for small RNA of prokaryote
  • High throughput library construction method for small RNA of prokaryote

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 15

[0027] Example 1 Construction of Fifteen Anaerobic Bacteria Prokaryotic Small RNA Chain Specific Libraries

[0028] 1. Removal of DNA from total RNA

[0029] 1. Use the following 28μl reaction system for DNA removal reaction:

[0030]

[0031] 2. Mix well, centrifuge briefly and incubate at 37°C for 15 minutes.

[0032] 2. Removal of rRNA in RNA samples after DNA removal

[0033] Use Illumina's Ribo-Zero kit to remove rRNA, the steps are as follows:

[0034] 1. RiboZero TM Magnetic bead preparation

[0035] A. Vortex and mix the magnetic beads that have been equilibrated at room temperature for 30 minutes, take 225 μl to a 1.5ml centrifuge tube with low adsorption and no RNase, place on the magnetic stand for 2 minutes until the supernatant is clear, and discard the supernatant.

[0036] B. Remove the centrifuge tube from the magnetic stand, add 225 μL of RNase-free water equilibrated at room temperature, blow and aspirate at the bottom of the tube ten times to mix, pl...

Embodiment 2 3

[0110] Example 2 Construction of three Mycobacterium smegmatis small RNA high-throughput sequencing libraries

[0111] 1. Removal of DNA from total RNA

[0112] 1. Use the following 28μl reaction system for DNA removal reaction:

[0113]

[0114] 2. Pipet and mix the above 28 μl reaction system, centrifuge briefly, and incubate at 37°C for 15 minutes.

[0115] 2. Removal of rRNA in RNA samples after DNA removal

[0116] Use Illumina's Ribo-Zero kit to remove rRNA, the steps are as follows:

[0117] 1. RiboZero TM Magnetic bead preparation

[0118] A. Vortex and mix the magnetic beads that have been equilibrated at room temperature for 30 minutes, take 225 μl to a 1.5ml centrifuge tube with low adsorption and no RNase, place on the magnetic stand for 2 minutes until the supernatant is clear, and discard the supernatant.

[0119] B. Remove the centrifuge tube from the magnetic stand, add 225 μL of RNase-free water equilibrated at room temperature, blow and aspirate at th...

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Abstract

The invention provides a high throughput library construction method for small RNA of prokaryote. The construction method includes the following steps of 1, digesting DNA in total RNA; 2, removing rRNA; 3, using random primers to synthesize a first chain of cDNA; 4, synthesizing a second chain of the cDNA; 5, screening the small RNA of the prokaryote in an electrophoresis mode through cutting gel; 6, repairing the tail end, adding A, carrying out purifying after linker adding, carrying out PCR amplification on a purified product, purifying a computer library, and constructing the computer library. According to the high throughput library construction method, after separation of the small RNA of the prokaryote is adjusted to the fact that the two chains are combined and converted into the cDNA, RNA degradation caused by separation on the RNA level can be avoided, the proportion of rRNA in sequencing data is greatly reduced, and the complete sequence of the small RNA of the prokaryote is obtained.

Description

technical field [0001] The present invention relates to the field of biotechnology, specifically, the present invention relates to the field of high-throughput sequencing library construction, and more specifically, the present invention relates to a high-throughput sequencing library construction method for prokaryotic small RNA. Background technique [0002] Small RNA is a general term for a class of non-coding short RNAs with important regulatory functions in organisms, mainly including miRNA, piRNA and siRNA. A large number of studies have confirmed that small RNAs are involved in the regulation of almost all life processes of organisms, including cell proliferation, differentiation, apoptosis and so on. Small RNA sequencing is to analyze this type of important regulatory small RNA, using high-throughput sequencing technology to identify and analyze small RNA sequences in samples. [0003] In eukaryotes, small RNAs exist in longer RNA transcripts in a stem-loop structur...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12N15/10
Inventor 郑洪坤关宁方涛
Owner BIOMARKER TECH
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