A high-throughput single-cell small RNA library construction method

Abstract

The invention provides a method for constructing a high-throughput single-cell small RNA library. The method is as follows: prepare a single cell suspension, add it into the microwell of the chip of the single cell operating system, and select the microwell of a single living cell to conduct the experiment; perform cell lysis reaction, 3' end connection reaction, and removal of free cells in sequence. Adapter reaction, 5' end ligation reaction, reverse transcription reaction and two PCR reactions; products are purified and fragment screening recovered to obtain a single-cell small RNA library that can be directly used for sequencing on the machine; 3' end ligation reaction During the process, the nucleotide sequence of the 3' linker used is shown in SEQ ID NO: 1; during the 5' end ligation reaction, the nucleotide sequence of the 5' linker used is shown in SEQ ID NO: 2 shown. The method of the invention has the advantages of high accuracy, high sensitivity, good repeatability and the like.

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 kind of non-coding RNA that widely exists 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 repression, mRNA splicing and deadenylation, and plays an important role in regulating gene expression, cell cycle, and organism 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 evaluation of treatment response. [0003] In the prior art, biological companies such as Illumina and NEB use small RNA standard library construction kits. The sample requires a high initial amoun...

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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