Construction method of single cell RNA sequencing library

A technology of sequencing library and construction method, applied in the field of single-cell RNA sequencing, can solve the problems of inability to obtain full-length transcript chain information of multiple transcript RNAs, unrealistic expression of gene expression, and unsatisfactory conditions, and reduce sequencing. Cost, comprehensive coverage, cost reduction effect

Active Publication Date: 2019-08-23
ZHEJIANG UNIV
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Problems solved by technology

[0009] However, at present, this technology still has the following deficiencies: this technology is only for the construction of mRNA libraries, and pre-amplification of full-length cDNA will inevitably lead to untrue expression of g

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  • Construction method of single cell RNA sequencing library
  • Construction method of single cell RNA sequencing library
  • Construction method of single cell RNA sequencing library

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preparation example Construction

[0063] Preparation of carrier RNA

[0064] The corresponding DNA fragments of the carrier RNA containing the 3' end poly A tail and the carrier RNA sequence not containing the 3' end poly A tail can add T7 promoter sequence at the 5' end (Schenborn et al., 1985 ) into the carrier (bacterial plasmid), the complete DNA sequence containing the T7 promoter can be double-digested through the restriction enzyme site on the carrier, and the DNA fragments are separated by agarose gel electrophoresis, and the gel is recovered to obtain the target fragment. The target DNA fragment obtained after purification was obtained by in vitro transcription kit (HiScribe T7 Quick High Yield RNA Synthesis Kit, NEB#2050) to obtain carrier RNA, carrier RNA larger than 200 bases can be obtained by PlusMini Kit (Qiagen) for purification, the carrier RNA less than 200 bases can be purified by MEGAclear TM KitPurification for Large Scale Transcription Reactions (Ambion) for purification, smallcarrier...

Embodiment 1

[0066] Example 1 Poly (A) selected single-cell RNA library construction (see the attached flowchart for details figure 2 )

[0067] 1.1 Obtain single cells by micromanipulation or flow cytometry for lysis. The method of lysis can be heated at 95°C for 5 minutes in 0.1% BSA / PBS or other non-ionic surfactants. Add about 100ng of carrier RNA, the carrier RNA here is a mixed sample, 5%-10% of which is a carrier RNA (SEQ ID NO.:1) containing a 3'-end polyadenylic acid tail, and the rest is a carrier RNA that does not contain a 3'-end polyadenylic acid tail Adenylate-tailed carrier RNA (SEQID NO.:2), the final volume is 25 μL, and the insufficient part can be made up with nuclease-free water.

[0068] The next process of building a library starts with Ultra TM RNA Library Prep Kit for Illumina (#E7530) kit method as an example:

[0069] 1.2 by NEBNext Oligo d(T) 25 10 μL of magnetic beads to obtain RNA containing Poly(A), Poly(A) mRNAMagnetic Isolation Module (NEB#E7490)

[...

Embodiment 2

[0117] Example 2 Non-Poly(A)-selected chain-specific single-cell RNA library construction (see the attached flowchart for details image 3 )

[0118] 2.1 Obtain single cells by micromanipulation or flow cytometry for lysis. The method of lysis is to heat at 95°C for 5 minutes in 0.1% BSA / PBS or a lysate prepared with other non-ionic surfactants, and add about 100ng of carrier at the same time RNA, the carrier RNA here is the carrier RNA (SEQ ID NO.: 2) that does not contain the 3'-end polyadenylic acid tail, the final system is 3 μL, and the insufficient part can be made up with nuclease-free water.

[0119] The next process of building a library starts with Ultra TM Directional RNA Library Prep Kit for (#E7420) kit method as an example:

[0120] 2.2 Fragmentation of RNA and random primer binding

[0121]

[0122] Mix well, with a total volume of 12 μL, 94°C, 15 minutes, fragment the RNA to a size of about 200 bases (the adjustment of the fragment size can change th...

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Abstract

The invention relates to the field of biotechnology and provides a construction method of a single-cell RNA sequencing library. The method includes the following steps that 1, carrier RNA is added ina single cell lysis system to ensure the RNA quantity required by library construction without pre-amplification of full-length cDNA; 2, the library is constructed by using a commercial kit or RNA sequencing library construction method; 3, DNA formed through carrier RNA transcription is removed from the library; 4, high-throughput sequencing is performed. The pre-amplification step of the full-length cDNA in an existing method is omitted, in the library construction process, the added RNA with the known sequence is subjected to digestion removal and prevented from causing interference to sequencing, and the sequencing cost is greatly reduced.

Description

technical field [0001] The invention relates to the field of single-cell RNA sequencing, including single-cell transcriptome RNA, smallRNA, and materials and methods for constructing RNA strand-specific sequencing libraries. Background technique [0002] In the past, microarray (Microarray) was used to obtain gene expression profiles. With the development of next-generation sequencing technology, RNA-Seq is also because of its high throughput, accurate quantification, high reproducibility, and can be used for isoform and Features such as single nucleotide polymorphism site (SNP) detection are gradually being widely used in scientific and clinical research, and most of these methods are based on the premise of obtaining a lot of cells and a large amount of RNA. The reality is that these methods do not reveal differences in gene expression in individual cells, but only reflect the absolute amount of gene expression in a multicellular ensemble. [0003] In many cases, the numb...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869C40B50/06
CPCC12Q1/6869C40B50/06C12Q2535/122
Inventor 贾俊岭肖政云程果吴超
Owner ZHEJIANG UNIV
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