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Second-generation sequencing technology based microbe unicell transcriptome analysis method

A next-generation sequencing technology and transcriptome analysis technology, which is applied in the field of microbial single-cell transcriptome analysis based on next-generation sequencing technology, and can solve problems such as the inability to reveal metabolic function gene expression kinetic data.

Active Publication Date: 2015-04-29
TIANJIN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although single-cell genomics technology has been successfully implemented, because it only provides information on the genetic structure and metabolic potential of cells, it cannot reveal the expression kinetics data of genes related to metabolic functions and environmental parameters.

Method used

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  • Second-generation sequencing technology based microbe unicell transcriptome analysis method
  • Second-generation sequencing technology based microbe unicell transcriptome analysis method
  • Second-generation sequencing technology based microbe unicell transcriptome analysis method

Examples

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Embodiment 1

[0029] In this embodiment, the prokaryotic microorganism cyanobacteria Synechocystis sp. PCC6803 was selected as the experimental material. The transcriptome of the cells (24 and 72 hours of nitrogen starvation) and the transcriptome of the population cells at the corresponding time were analyzed. A microbial single-cell transcriptome analysis method based on next-generation sequencing technology, comprising the following steps:

[0030] (1) Separation of microbial single cells:

[0031] The fresh microbial cyanobacteria Synechocystis sp. PCC6803 cell culture samples were collected by centrifugation at room temperature, immediately suspended in the RNA protection agent RNALater solution and fully resuspended; using NARISHIGE IM-9C microinjection system (Narishige, Tokyo , JP) Under the Olympus IX71 inverted microscope, single cells were selected and isolated, and the isolated microbial single cells were resuspended in RNALater solution;

[0032] (2) Extraction of total RNA: ...

Embodiment 2

[0058] Quality Control of cDNA Libraries

[0059] Ligate, transform, and clone the cDNA library obtained in step (3) of Example 1, and randomly select clones for sequencing:

[0060] 1) Use the End-It DNA End-Repair Kit kit from Epicentre, USA to complete the end of the cDNA library, and then use CLONESMART Blunt Cloning Kits to combine the purified cDNA library with 10X End-Repair Buffer, dNTP, ATP, End-Repair Mix the Enzyme Mix evenly, incubate at room temperature for 45 minutes, then incubate at 70°C for 10 minutes to terminate the reaction.

[0061] 2) Use CLONESMART Blunt Cloning Kits to mix the end-filled cDNA library with 4X CloneSmart VectorPremix and CloneSmart DNA Ligase, incubate at room temperature for 30 minutes, then transform into Escherichia coli to construct a clone library, and randomly select single clones from the library for sequencing analyze.

Embodiment 3

[0063] Quantification of RNA-Seq Libraries

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Abstract

The invention discloses a second-generation sequencing technology based microbe unicell transcriptome analysis method. The analysis method comprises the following steps: (1) separating microbe unicells; (2) extracting total RNA; (3) conducting linear amplification on total RNA; (4) constructing an RNA sequencing library; (5) analyzing the RNA sequencing library. By adopting the analysis method, analysis on gene expression difference among different microbe cells can be realized, and physiological function analysis of the unicell level on microbes which cannot be cultivated in extreme environments can be realized; furthermore, the analysis method can be suitable for analysis on eukaryotic unicells, and has quite wide applications in the fields of important biological and medical researches and the like, such as tumor generation and evolution, and stem cell induction and development.

Description

technical field [0001] The present invention relates to a method for analyzing molecular biology and transcriptomics, in particular to a method for analyzing microbial single-cell transcriptome based on next-generation sequencing technology. Background technique [0002] Non-culture-based single-cell genomics techniques have been successfully implemented and applied to various microbial ecology studies. The difficulty in analyzing microbial cells is that most microorganisms, such as bacteria and archaeal cells, have a cell outer membrane structure that is difficult to effectively lyse; prokaryotic microorganisms have short RNA half-lives and low stability; in addition, the cell size is much smaller than that of mammals Cells (10-30 picograms), so the total concentration of RNA molecules in cells is very low, about 4-10 Ficks per cell, this RNA concentration is much lower than the RNA concentration in eukaryotic cells, which is RNA Extraction and analysis present many challe...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6869C12Q2563/143C12Q2531/101
Inventor 王江新陈磊张卫文肖华志方文质李岩
Owner TIANJIN UNIV
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