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Single cell transcriptome sequencing method and application thereof

A transcriptome sequencing and single-cell technology, applied in biochemical equipment and methods, combinatorial chemistry, microbial measurement/inspection, etc., can solve the problems of low sensitivity, undetectable, poor sequencing effect, etc., and achieve RNA detection sensitivity high effect

Pending Publication Date: 2022-05-17
ZHEJIANG UNIV
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AI Technical Summary

Problems solved by technology

[0005] The reverse transcription primers used in the high-throughput single-cell transcriptome sequencing technology represented by 10X Genomics are all Poly(dT) reverse transcription primers, so only part of the transcript information at the 3' end can be obtained, and undesired transcripts cannot be detected. RNA containing polyA (including damaged mRNA fragments and polyA-free miRNA, lncRNA, etc.), which leads to low sensitivity of this technology in practical applications, usually less than 10% of mRNA can be detected
At the same time, this technology has high requirements on RNA quality. If you want to obtain better sequencing results, the cell viability of the sequencing sample is greater than 80%, and the sequencing effect is often poor when using frozen or fixed samples.
In addition, because bacterial RNA does not carry polyA, existing high-throughput single-cell transcriptome sequencing technologies cannot be used for bacterial transcriptome sequencing

Method used

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  • Single cell transcriptome sequencing method and application thereof
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  • Single cell transcriptome sequencing method and application thereof

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specific Embodiment approach

[0056] In order to make the purposes, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments These are some embodiments of the present invention, but not all embodiments.

[0057] Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present invention.

[0058] The technical solutions of the present invention are further described below with reference to the accompanying drawings and through specific embodiments.

Embodiment 1

[0059] Example 1: Sequencing of E. coli samples

[0060] sample preprocessing

[0061] Take about 1 million different strains (1, 2, 3, 4) of Escherichia coli to verify that the patent of the present invention can be used for single-cell transcriptome sequencing of bacterial samples. ) PBS buffer (PBST) was washed three times, and the agglomerated bacteria were removed by shaking and filtration, so that the bacteria in the sample formed a single bacterial suspension, and 1% paraformaldehyde (purchased from Beijing Soleibao Technology Co., Ltd.) was added. Fix overnight at 4°C. The fixed bacterial samples were washed three times with PBST buffer, lysozyme (purchased from Thermo Fisher Scientific, USA) was added to lyse the cell wall, treated at 37°C for 15 minutes, and washed three times by adding PBST buffer.

[0062] Reverse Transcription

[0063] The pretreated samples were added with reverse transcriptase (purchased from Thermo Fisher Scientific, USA), reverse tran...

Embodiment 2

[0073] Example 2: Sequencing of mixed samples of Escherichia coli and Bacillus subtilis

[0074] About 1 million mixed samples of Escherichia coli and Bacillus subtilis were taken to verify the accuracy of single-cell transcriptome sequencing using the patent of the present invention. Sample pretreatment, reverse transcription, addition of capture adapters, single cell isolation, synthesis of the second strand of cDNA, library construction, and high-throughput sequencing were performed using the methods described in Example 1 above. The sequencing results showed that a total of 251 bacteria were detected in the mixed samples of Escherichia coli and Bacillus subtilis after genome alignment, and 4 of them contained both Escherichia coli cDNA and Bacillus subtilis cDNA. The remaining bacteria contain almost only Escherichia coli cDNA or Bacillus subtilis cDNA, and the overall contamination rate is probably less than 2% (see Figure 5 ). It shows that the application of the pa...

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Abstract

The invention provides a single-cell transcriptome sequencing method and application thereof. The method comprises the following steps: preparing a to-be-detected cell sample into a single-cell suspension, and fixing the single-cell suspension with a stationary liquid; and performing an in-situ reverse transcription reaction on the RNA of the fixed single cell by using a reverse transcription primer to synthesize a cDNA first chain.

Description

Technical field [0001] The invention belongs to the field of biotechnology, relates to the field of single cell sequencing, and specifically relates to a single cell transcriptome sequencing method and its application. Background technology [0002] The most widely used high-throughput single-cell transcriptome sequencing technology at present is the single-cell sequencing technology based on the droplet microfluidic platform developed by 10X Genomics. This technology can realize the labeling, sequencing and analysis of thousands of cells, and obtain single-cell sequencing technology. Gene expression profiling at the cellular level enables the division of cell subpopulations and the detection of differentially expressed genes between cell subpopulations. Similar technologies include inDrop technology and Drop-seq technology. [0003] The basic operations and principles of 10X Genomics transcriptome sequencing technology are (see Figure 1 ): Prepare the sample into a single ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6869C40B50/06
CPCC12Q1/6806C12Q1/6869C40B50/06C12Q2525/191C12Q2543/101C12Q2531/113C12Q2535/122
Inventor 王永成徐子叶王钰婷
Owner ZHEJIANG UNIV
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