阅读说明：本技术 一种获得单细胞mRNA序列的方法 (Method for obtaining single-cell mRNA sequence ) 是由 范飞 程小芳 章文蔚 汪为茂 崔路漫 王欧 于 2018-12-27 设计创作，主要内容包括：本发明公开了一种获得单细胞mRNA序列的方法。本发明的方法：(1)利用细胞标签载体捕获细胞的mRNA,反转录得到具有细胞标签的cDNA；来自于同一个细胞的cDNA具有相同的细胞标签,来自于不同细胞的cDNA具有不同的细胞标签；(2)利用转座酶复合体和分子标签载体,得到具有分子标签的多个cDNA片段；来自于同一cDNA的各个片段具有相同的分子标签,来自于不同cDNA的片段具有不同的分子标签；(3)高通量测序；(4)根据分子标签进行序列拼接,得到每个mRNA的序列；根据细胞标签,得到每个单细胞的所有mRNA的序列。本发明提供的方法可用于高通量获得大量单细胞中每个单细胞的所有mRNA的序列。(The invention discloses a method for obtaining a single-cell mRNA sequence. The method comprises the following steps: (1) capturing mRNA of cells by using a cell tag carrier, and performing reverse transcription to obtain cDNA with a cell tag; cDNAs from the same cell have the same cell tag, and cDNAs from different cells have different cell tags; (2) utilizing the transposase complex and the molecular tag carrier to obtain a plurality of cDNA fragments with molecular tags; each fragment from the same cDNA has the same molecular tag, and fragments from different cdnas have different molecular tags; (3) high-throughput sequencing; (4) performing sequence splicing according to the molecular tags to obtain a sequence of each mRNA; from the cell tags, the sequences of all mrnas were obtained for each single cell. The method provided by the invention can be used for obtaining all mRNA sequences of each single cell in a large number of single cells at high flux.)

A method of obtaining a single cell mRNA sequence, comprising the steps of:

(1) capturing mRNA of cells by using a cell tag carrier, and then carrying out reverse transcription to obtain cDNA with a cell tag; the cell label carrier is a solid phase carrier carrying a cell label; cDNAs from the same cell have the same cell tag, and cDNAs from different cells have different cell tags;

(2) utilizing the transposase complex and the molecular tag carrier to obtain a plurality of cDNA fragments with molecular tags; each fragment from the same cDNA has the same molecular tag, and fragments from different cdnas have different molecular tags; the molecular label carrier is a solid phase carrier carrying a molecular label;

(3) high-throughput sequencing;

(4) performing sequence splicing on the sequencing result according to the molecular label to obtain a sequence of each mRNA; further based on the cell tags, the sequences of all mrnas were obtained for each single cell.

The method of claim 1, wherein:

the cell label carrier is obtained by connecting a plurality of DNA molecules B to the surface of a solid phase carrier; the DNA molecule B is a partial double-stranded structure and consists of two partially complementary single-stranded DNA molecules; the DNA molecule B has a cell label and an mRNA capture region.

The method of claim 1, wherein:

the molecular label carrier is obtained by connecting a plurality of DNA molecules A to the surface of a solid phase carrier; the DNA molecule A is a partial double-stranded structure and consists of two partially complementary single-stranded DNA molecules; the DNA molecule A is provided with a molecular label and a transposase complex capture region.

A method as claimed in claim 1, 2 or 3, characterized by:

a step of enriching the target object is included between the step (1) and the step (2);

and/or

A step of enriching the target substance is included between the step (2) and the step (3).

The method of claim 4, wherein:

and (3) the step of enriching the target object between the step (1) and the step (2) is PCR amplification or rolling circle amplification.

A method for obtaining and quantifying single cell mRNA sequences, comprising the steps of:

(1) capturing mRNA of cells by using a cell tag carrier, and then carrying out reverse transcription to obtain cDNA with a cell tag and a transcript tag; the cell label carrier is a solid phase carrier carrying a cell label and a transcript label; cDNAs from the same cell have the same cell tag, and cDNAs from different cells have different cell tags; each cDNA from the same cell has a different transcript tag;

(2) utilizing the transposase complex and the molecular tag carrier to obtain a plurality of cDNA fragments with molecular tags; each fragment from the same cDNA has the same molecular tag, and fragments from different cdnas have different molecular tags; the molecular label carrier is a solid phase carrier carrying a molecular label;

(3) high-throughput sequencing;

(4) performing sequence splicing on the sequencing result according to the molecular label to obtain each mRNA sequence; further obtaining the sequences of all mRNAs of each single cell according to the cell label; finally, the mRNA of each single cell is quantified according to the number of sequences with the same mRNA sequence but different transcript labels.

The method of claim 6, wherein:

the cell label carrier is obtained by connecting a plurality of DNA molecules B to the surface of a solid phase carrier; the DNA molecule B is a partial double-stranded structure and consists of two partially complementary single-stranded DNA molecules; the DNA molecule B is provided with a cell label, a transcript label and an mRNA capture region.

The method of claim 6, wherein:

the molecular label carrier is obtained by connecting a plurality of DNA molecules A to the surface of a solid phase carrier; the DNA molecule A is a partial double-stranded structure and consists of two partially complementary single-stranded DNA molecules; the DNA molecule A is provided with a molecular label and a transposase complex capture region.

The method of claim 6, 7 or 8, wherein:

a step of enriching the target object is included between the step (1) and the step (2);

and/or

A step of enriching the target substance is included between the step (2) and the step (3).

The method of claim 9, wherein:

and (3) the step of enriching the target object between the step (1) and the step (2) is PCR amplification or rolling circle amplification.

A method of preparing a single cell cDNA sequencing library comprising the steps of:

(1) obtaining cDNA with a cell tag; cDNAs from the same cell have the same cell tag, and cDNAs from different cells have different cell tags;

(2) obtaining a plurality of cDNA fragments with molecular tags; each fragment from the same cDNA has the same molecular tag, and fragments from different cDNAs have different molecular tags.

The method of claim 11, wherein: the method for obtaining cDNA with cell tag is as follows: capturing mRNA of cells by using a cell tag carrier, and then carrying out reverse transcription to obtain cDNA with a cell tag; the cell label carrier is a solid phase carrier carrying a cell label.

The method of claim 11, wherein: the method for obtaining a plurality of cDNA fragments with molecular tags is realized as follows: utilizing the transposase complex and the molecular tag carrier to obtain a plurality of cDNA fragments with molecular tags; the molecular label carrier is a solid phase carrier carrying molecular labels.

The method of claim 12, wherein:

the cell label carrier is obtained by connecting a plurality of DNA molecules B to the surface of a solid phase carrier; the DNA molecule B is a partial double-stranded structure and consists of two partially complementary single-stranded DNA molecules; the DNA molecule B has a cell label and an mRNA capture region.

The method of claim 13, wherein:

the molecular label carrier is obtained by connecting a plurality of DNA molecules A to the surface of a solid phase carrier; the DNA molecule A is a partial double-stranded structure and consists of two partially complementary single-stranded DNA molecules; the DNA molecule A is provided with a molecular label and a transposase complex capture region.

The method of claim 11 or 12 or 13 or 14 or 15, wherein:

the step of enriching the target substance is included between the step (1) and the step (2).

The method of claim 16, wherein:

the step of enriching the target is PCR amplification or rolling circle amplification.

A method for preparing a single cell quantitative cDNA sequencing library, comprising the steps of:

(1) obtaining a cDNA having a cell tag and a transcript tag; cDNAs from the same cell have the same cell tag, and cDNAs from different cells have different cell tags; each cDNA from the same cell has a different transcript tag;

(2) obtaining a plurality of cDNA fragments with molecular tags; each fragment from the same cDNA has the same molecular tag, and fragments from different cDNAs have different molecular tags.

The method of claim 18, wherein: the method for obtaining cDNA with cell tag and transcript tag is as follows: capturing mRNA of cells by using a cell tag carrier, and then carrying out reverse transcription to obtain cDNA with a cell tag and a transcript tag; the cell label carrier is a solid phase carrier carrying a cell label and a transcript label.

The method of claim 18, wherein: the method for obtaining a plurality of cDNA fragments with molecular tags is realized as follows: utilizing the transposase complex and the molecular tag carrier to obtain a plurality of cDNA fragments with molecular tags; the molecular label carrier is a solid phase carrier carrying molecular labels.

The method of claim 19, wherein:

the cell label carrier is obtained by connecting a plurality of DNA molecules B to the surface of a solid phase carrier; the DNA molecule B is a partial double-stranded structure and consists of two partially complementary single-stranded DNA molecules; the DNA molecule B is provided with a cell label, a transcript label and an mRNA capture region.

The method of claim 20, wherein:

the molecular label carrier is obtained by connecting a plurality of DNA molecules A to the surface of a solid phase carrier; the DNA molecule A is a partial double-stranded structure and consists of two partially complementary single-stranded DNA molecules; the DNA molecule A is provided with a molecular label and a transposase complex capture region.

The method of claim 18 or 19 or 20 or 21 or 22, wherein:

the step of enriching the target substance is included between the step (1) and the step (2).

The method of claim 23, wherein:

the step of enriching the target is PCR amplification or rolling circle amplification.

A kit for preparing a single cell cDNA sequencing library comprises a cell tag carrier and a molecular tag carrier; the cell label carrier is a solid phase carrier carrying a cell label and is used for obtaining cDNA with the cell label, the cDNA from the same cell has the same cell label, and the cDNA from different cells has different cell labels; the molecular tag carrier is a solid phase carrier carrying molecular tags and is used for obtaining a plurality of cDNA fragments with molecular tags, wherein each fragment from the same cDNA has the same molecular tag, and the fragments from different cDNAs have different molecular tags.

The kit of claim 25, wherein:

the cell label carrier is obtained by connecting a plurality of DNA molecules B to the surface of a solid phase carrier; the DNA molecule B is a partial double-stranded structure and consists of two partially complementary single-stranded DNA molecules; the DNA molecule B has a cell label and an mRNA capture region.

The kit of claim 25, wherein:

the molecular label carrier is obtained by connecting a plurality of DNA molecules A to the surface of a solid phase carrier; the DNA molecule A is a partial double-stranded structure and consists of two partially complementary single-stranded DNA molecules; the DNA molecule A is provided with a molecular label and a transposase complex capture region.

The kit of claim 25 or 26 or 27, wherein: the kit also includes a transposase complex.

The kit of claim 28, wherein: the transposase complex is composed of a transposon and a transposase.

A kit for preparing a single-cell quantitative cDNA sequencing library comprises a cell label carrier and a molecular label carrier; the cell label carrier is a solid phase carrier carrying a cell label and a transcript label and is used for obtaining cDNA with the cell label and the transcript label, the cDNA from the same cell has the same cell label, the cDNA from different cells has different cell labels, and each cDNA from the same cell has different transcript labels; the molecular tag carrier is a solid phase carrier carrying molecular tags and is used for obtaining a plurality of cDNA fragments with molecular tags, wherein each fragment from the same cDNA has the same molecular tag, and the fragments from different cDNAs have different molecular tags.

The kit of claim 30, wherein:

the cell label carrier is obtained by connecting a plurality of DNA molecules B to the surface of a solid phase carrier; the DNA molecule B is a partial double-stranded structure and consists of two partially complementary single-stranded DNA molecules; the DNA molecule B is provided with a cell label, a transcript label and an mRNA capture region.

The kit of claim 30, wherein:

the molecular label carrier is obtained by connecting a plurality of DNA molecules A to the surface of a solid phase carrier; the DNA molecule A is a partial double-stranded structure and consists of two partially complementary single-stranded DNA molecules; the DNA molecule A is provided with a molecular label and a transposase complex capture region.

The kit of claim 30, 31 or 32, wherein: the kit also includes a transposase complex.

The kit of claim 33, wherein: the transposase complex is composed of a transposon and a transposase.

Use of the method of claim 1 or 2 or 3 or 4 or 5 or 11 or 12 or 13 or 14 or 15 or 16 or 17 for high throughput obtaining of all mRNA sequences of each of a plurality of single cells.

Use of the method of claim 6 or 7 or 8 or 9 or 10 or 18 or 19 or 20 or 21 or 22 or 23 or 24 for high throughput obtaining and quantifying the sequence of all the mrnas of each of a plurality of single cells.

Use of the kit of claim 25 or 26 or 27 or 28 or 29 for high throughput obtaining of the sequence of all mRNA of each of a plurality of single cells.

Use of the kit of claim 30 or 31 or 32 or 33 or 34 for high throughput obtaining and quantifying the sequence of all mRNA of each of a plurality of single cells.