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Magnetic microsphere with molecular tag sequence, and preparation method for magnetic microsphere

A technology of magnetic microspheres and molecular tags, which is applied in the field of molecular biology, can solve problems affecting the stability and accuracy of single-cell sequencing results, high cost of single-cell sequencing, and unstable batch results, so as to achieve convenient source of raw materials, The effect of low cost and small batch variance

Pending Publication Date: 2021-01-22
SINGLERON NANJING BIOTECHNOLOGIES LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, microbeads or magnetic beads with molecular tags currently have many problems such as high production cost, difficult preparation, low preparation success rate, and unstable results in different batches, which seriously affect the stability and accuracy of single-cell sequencing results. At the same time, this is also one of the reasons why single-cell sequencing is expensive and cannot be widely used.

Method used

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  • Magnetic microsphere with molecular tag sequence, and preparation method for magnetic microsphere
  • Magnetic microsphere with molecular tag sequence, and preparation method for magnetic microsphere
  • Magnetic microsphere with molecular tag sequence, and preparation method for magnetic microsphere

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1: the preparation of magnetic microsphere

[0033] (1) Oligo dissolved

[0034] The first stage of oligo dissolution: take 96 strips of R1 in a centrifuge, centrifuge at 12000rpm for 2min to make the R1 settle to the bottom, add an appropriate amount of 0.1M MES buffer to dissolve to a final concentration of 50μM; let stand at room temperature for 10min, and dissolve the above-dissolved R1 Vortex for 20 s, centrifuge clockwise in a handheld centrifuge to settle R1 at the bottom, and store R1 at 4°C for later use. If the frequency of use is not high, it can be stored at -20°C for later use.

[0035] Dissolving other oligos (R2, R3): Place R2 or R3 in a centrifuge, centrifuge at 12,000 rpm for 2 minutes to settle to the bottom, add an appropriate amount of 1×TE buffer to dissolve to a final concentration of 50 μM. After standing at room temperature for 10 minutes, vortex and shake for 20 seconds, spin in a handheld centrifuge to settle at the bottom and store a...

Embodiment 2

[0047] Embodiment 2: Performance analysis of the magnetic microspheres prepared by the present invention

[0048] In this embodiment, three groups are divided into three groups for single-cell mRNA sequencing to verify the beneficial performance of the prepared magnetic microspheres. For specific single-cell mRNA sequencing procedures, refer to the attached figure 1 shown. Among them, group D1 used dropseq microbeads commonly used in the market, and groups C1 and C2 were magnetic microspheres A and magnetic microspheres B prepared in Example 1, respectively. In this example, the same batch of single-cell sequencing kits was used for testing. Except for the different microspheres, other conditions remained the same throughout the test. Specific steps are as follows:

[0049] (1) Preparation of cells and massive single-cell mRNA sequencing pretreatment kits;

[0050] (2) Cooperate with Singleron Matrix automated single-cell sequencing library construction system to obtain sin...

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Abstract

The invention belongs to the field of the molecular biology, and particularly relates to a magnetic microsphere with a molecular tag sequence, and a preparation method for the magnetic microsphere. Amagnetic bead is taken as a molecular tag base, and a chemical bond and enzyme linking method is used for linking three-segment oligo to the magnetic bed in sequence so as to synthesize the magnetic microsphere with a unique oligo sequence. The magnetic microsphere prepared by the preparation method disclosed by the invention has high stability and accuracy. Meanwhile, the preparation technology of the magnetic microsphere is simple, time consumption is small, raw material sources are convenient, the cost is low, and the unicellular sequencing cost is greatly lowered. The oligo adopted by themagnetic microsphere is a known sequence, the three-segment oligo can be combined to form millions of molecular tags, batch differences are small, the stability and the accuracy of a unicellular sequencing result are obviously improved, and the magnetic microsphere can be widely applied to medical research, bioresearch and the like.

Description

technical field [0001] The invention belongs to the field of molecular biology, and in particular relates to a magnetic microsphere with a molecular tag sequence and a preparation method thereof. Background technique [0002] Single-cell transcriptome sequencing (single-cell RNA-sequencing, scRNA-seq) is a new technology that has emerged in recent years. It can obtain the expression profile of the whole transcriptome from the single cell level, perform high-throughput sequencing after amplification, and efficiently Detecting the gene expression level in a single cell has important application value in the development and differentiation of stem cells, diagnosis and treatment of tumors, and the design of targeted drugs. [0003] The reason why single-cell transcriptome sequencing can sequence hundreds of thousands of cells at the same time is that each cell has a different label. In order to distinguish each mRNA in each cell, each mRNA is also given a unique molecular label...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869B01J13/02
CPCC12Q1/6869B01J13/02C12Q2563/149C12Q2563/143
Inventor 杨柳訾晓渊
Owner SINGLERON NANJING BIOTECHNOLOGIES LTD
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