Method for establishing high-throughput single-cell small RNA (ribonucleic acid) library

Abstract

The invention provides a method for establishing a high-throughput single-cell small RNA (ribonucleic acid) library. The method comprises the following steps: preparing a single-cell suspension, adding the single-cell suspension to micropores of a chip of a single-cell operating system, and selecting micropores of single living cells for experiment; performing a cell lysis reaction, a 3'-end connection reaction, a free junction removal reaction, a 5'-end connection reaction, a reverse transcription reaction and two PCR (polymerase chain reaction) reactions successively; and performing productpurification and fragment screening recycling, so as to obtain a single-cell small RNA library, wherein the nucleotide sequence of 3'-junction used in the 3'-end connection reaction is shown in SEQ IDNO: 1 in the description; and the nucleotide sequence of 5'-junction used in the 5'-end connection reaction is shown in SEQ ID NO: 2 in the description. The method provided by the invention has the advantages of high accuracy, high sensitivity and good repeatability.

Description

Technical field [0001] The invention belongs to the technical field of single-cell small RNA sequencing, and relates to a method for constructing a high-throughput single-cell small RNA library. Background technique [0002] miRNA is a non-coding RNA that is widely present in eukaryotes and can regulate the expression of other genes. miRNA is partially complementary to one or more mRNA molecules, and down-regulates gene expression in various ways, including translational inhibition, mRNA splicing and deadenylation, and plays an important role in regulating gene expression, cell cycle, and biological development timing. . miRNA also plays a key role in the formation of cancer and other related diseases, and has been used in cancer diagnosis, staging, progression, prognosis, and assessment of treatment response. [0003] In the prior art, biological companies such as Illumina and NEB use small RNA standard library building kits. The sample requires a high starting amount, with a m...

AI Technical Summary

Problems solved by technology

The sample requires a high initial amount, with a minimum of 100ng total RNA, which cannot achieve the level of single cell (10pg / cell); without designing barcode, it is impossible to mix multiple samples to build a library; without designing UMI, it is impossible to reduce the preference introduced by PCR ;There are many self-ligation products and non-specific products of adapters, which lead to difficulties in ligation and amplification of target fragments, and low efficiency of library construction; PAGE gel recovery experiments are cumbersome and risky

Faridani et al. published in Nature biotechnology in 2016 and nature protocol in 2018 about the process of building a small RNA library in a single cell. Due to the excess adapter residues and the lack of screening and recovery of target fragments during the experiment, the linker in the product resulted in The self-linkage yield is extremely high, the content of the target product is less than 1%, and the amount of data required for sequencing is greatly increased, resulting in a great waste; the protocol does not have single-cell molecular labels, which makes it impossible to increase the throughput of the experiment, and cannot perform multiple cells at the same time Operation; this scheme is improved for the 3' adapter and 5' adapter without adding random sequences at the end, so that the ligation reaction may have a preference during the ligation reaction

[0004] Therefore, no feasible method has been developed to analyze single-cell miRNA expression profiles in highly heterogeneous tumor samples.

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