# US 2022/0033809 A1 · Method and kit for construction of rna library

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- Number: US 2022/0033809 A1
- Document ID: US20220033809A1
- Status: Published application
- Kind: A1
- Application number: 17/274,009
- Filed: 2018-09-05
- Published: 2022-02-03
- Earliest claimed: 2018-09-05
- Applicants: Bgi Shenzhen
- Inventors: Xi YANG; Wenjing WANG; Yanru XING; Jinjin XU; Fang CHEN; Shida ZHU
- Web page: https://patentgrep.com/patent/US20220033809A1

## CPC

- C12N 15/1072: {Differential gene expression library synthesis, e.g. subtracted libraries, differential screening}
- C12N 15/1096: {cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR}
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

Provided are an RNA library construction method and a specialized kit thereof. The method for preparing an RNA library includes the following steps: extracting RNA and performing fragmentation; adding a tail to 3′ end; ligating an adaptor to 5′ end and hybridizing with a DNA probe mixture; the DNA probe mixture is composed of several DNA probes that are reverse complementary to an RNA that is expected to be removed; removing RNA from the hybrid and removing DNA; performing reverse transcription and PCR amplification to obtain a library solution. The present invention combines polyA tailing and 5′end ligation. The RNA content in the system can be quickly increased by polyA tailing, thereby avoiding subsequent purification losses.

## Claims

### Claim 1

A method for preparing an RNA library, comprising the following steps:
(i) (a) extracting RNA and performing fragmentation; or (b) extracting RNA;
(ii) adding a tail to 3′ end;
(iii) ligating an adaptor to 5′ end, and hybridizing with a DNA probe mixture; the DNA probe mixture is composed of several DNA probes that are reverse complementary to an RNA that is expected to be removed;
(iv) removing RNA from the hybrid and removing DNA;
(v) performing reverse transcription and PCR amplification to obtain a library solution.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the fragmentation is implemented by heat treatment.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the step of adding a tail to 3′ end is implemented by the following manner: modifying an RNA fragment to have a phosphate group at its 5′ end and a hydroxyl group at its 3′ end, and then adding a tail.

### Claim 4 (depends on 3)

The method according to claim 3, wherein: the step of “modifying an RNA fragment to have a phosphate group at its 5′ end and a hydroxyl group at its 3′ end” is implemented by the following method: treating with T4 PNK.

### Claim 5 (depends on 1)

The method according to claim 1, wherein the step of removing RNA from the hybrid and removing DNA is implemented by the following method: performing RNaseH digestion and DNase digestion sequentially.

### Claim at position 6

6-7. (canceled)

### Claim 8

A method for preparing an RNA library, comprising the following steps:
(i) (a) extracting RNA and performing fragmentation; or (b) extracting RNA;
(ii) adding a tail to 3′ end;
(iii) ligating an adaptor to 5′ end and enriching a target RNA fragment;
(iv) performing reverse transcription and PCR amplification to obtain a library solution.

### Claim 9

(canceled)

### Claim 10

A kit for preparing an RNA library, comprising
(1) a component A, a component B, a component C, a component D, a component E, and a component F;
(2) a component A, a component G, a component D, a component E, and a component F;
(3) a component A, a component H, a component B, a component C, a component D, a component E, and a component F;
(4) a component A, a component H, a component G, a component D, a component E, and a component F;
(5) a component I, a component B, a component C, a component D, a component E, and a component F;
or,
(6) a component I, a component G, a component D, a component E, and a component F;
wherein:
Component A is a reagent or a combination of reagents for adding a tail to the 3′ end of an RNA molecule;
Component B is a reagent or a combination of reagents for ligating an adaptor to the 5′ end of an RNA molecule;
Component C is a DNA probe mixture; the DNA probe mixture is composed of several DNA probes that are reverse complementary to an RNA that is expected to be removed;
Component D is a reagent or a combination of reagents for removing RNA from the hybrid and removing DNA;
Component E is a reagent or a combination of reagents for reverse transcription;
Component F is a reagent or a combination of reagents for PCR;
Component G is a reagent or a combination of reagents for ligating an adaptor to the 5′ end of an RNA molecule and for hybridizing with a DNA probe;
Component H is a reagent or a combination of reagents for modifying an RNA fragment to have a phosphate group at its 5′ end and a hydroxyl group at its 3′ end;
Component I is a reagent or a combination of reagents for tailing and modifying the 3′ end of an RNA molecule.

### Claim at position 10

11-18. (canceled)

### Claim 19 (depends on 1)

The method of claim 1, wherein the method is for sequencing.

### Claim 20 (depends on 8)

The method of claim 8, wherein the method is for sequencing.