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

A library construction and single-cell technology, applied in libraries, chemical libraries, nucleotide libraries, etc., can solve the problems of ligation reaction preference, inability to mix multiple samples to build a library, and high output of adapter self-ligation

Active Publication Date: 2020-02-04
NAT INST OF BIOLOGICAL SCI BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0302] Example 1 High-throughput single-cell small RNA library construction of A549 cells (flow chart is shown in figure 1 Shown)

[0303] (1) Prepare A549 cells

[0304] 10% (v / v) fetal bovine serum and 1% penicillin-streptomycin were added to the DMEM / F-12 minimal medium as the culture environment for the A549 cell line. The A549 cell line was cultured in a humidified incubator with an ambient temperature of 37°C and a carbon dioxide content of 5%. In the experiment, fresh cells were taken, washed twice with 1X phosphate buffered saline (DPBS) water, and then suspended in 1X DPBS containing 0.04% bovine serum albumin. The cell suspension was stained with 4',6-diamidino-2-phenylindole (DIPA) to mark dead cells. The live cells were sorted and enriched with a BD FACSAria III flow cytometer to obtain a cell suspension.

[0305] (2) Stain and count the cells to prepare a single cell suspension

[0306] The cell suspension was stained with Hoechst-33342 and propidium iodide, and place...

Embodiment 2

[0319] Example 2 Construction of a high-throughput single cell small RNA library of human peripheral blood mononuclear cells (PBMCs)

[0320] (1) Preparation of human peripheral blood mononuclear cells

[0321] The venous blood of healthy blood donors was collected into a collection tube containing heparin sodium anticoagulant, and peripheral blood mononuclear cells (PBMCs) were separated by Ficoll density gradient centrifugation. The heparinized blood was dissolved with an equal volume of 1x DPBS and added to a SepMateTM-15 separation tube containing an equal volume of HISTOPAQUE-1077. After centrifuging the separation tube at a centrifugal force of 1200xg for 10 minutes, quickly pour the PBMCs into a new 50ml tube, wash twice with 1xDPBS, and then suspend the cells in 1xDPBS. The cells were stained with CD45, CD3, CD19+, CD20, CD56+, CD14+, and sorted and enriched with a BD FACSAria III flow cytometer.

[0322] The high-throughput single-cell small RNA library was constructed on ...

Embodiment 3

[0323] Example 3 Construction of a high-throughput single-cell small RNA library of mouse B16F10 cells

[0324] (1) Cultivation of mouse B16F10 cells and tumor implantation

[0325] 10% (v / v) fetal bovine serum and 1% penicillin-streptomycin were added to DMEM minimal medium as the culture environment for the melanoma cell line B16F10. B16F10 cells were cultured in a humidified incubator with an ambient temperature of 37°C and a carbon dioxide content of 5%.

[0326] Five C57bl / 6 female mice aged 8-10 weeks were free to eat and drink under SPF-free conditions. The mice were placed in a 12-hour light-dark cycle IVC cage and fed with Co60 radiation sterilized laboratory feed. On the day of the implantation experiment, change the fresh medium to culture for 4 hours and collect the cells. Suspend the cells in cold 1xDPBS to a final concentration of 3x10 5 cell / ml. Inject 100ul of cell suspension into the tail vein of each animal to produce experimental lung metastasis. From the 12th ...

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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...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/06
CPCC40B50/06C40B40/06
Inventor 蔡涛李佳
Owner NAT INST OF BIOLOGICAL SCI BEIJING
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