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Molecular marker micro-bead and high-throughput single cell sequencing method based on same

A single-cell sequencing and molecular labeling technology, applied in the field of high-throughput single-cell sequencing and molecular-labeled microbeads, can solve the problem that single-cell sorting cannot achieve high-throughput single-cell sequencing analysis, and achieve good application value, The effect of low experimental cost and high throughput

Active Publication Date: 2017-03-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, other manual picking, single cell sorting modified by machine and flow technology cannot meet the requirements of high-throughput single cell sequencing analysis

Method used

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  • Molecular marker micro-bead and high-throughput single cell sequencing method based on same
  • Molecular marker micro-bead and high-throughput single cell sequencing method based on same
  • Molecular marker micro-bead and high-throughput single cell sequencing method based on same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] 1) 1ng starting DNA fragmentation

[0048]Vazyme TD513 kit was used.

[0049] a. Thaw 5×TTBL (TruePrep Tagment Buffer L) at room temperature, invert and mix well before use. Confirm that 5×TS (Terminate Solution, reaction termination solution) is at room temperature, and flick the tube wall to confirm whether there is precipitation. If there is precipitation, heat at 37°C and shake vigorously to mix well and the precipitation will dissolve.

[0050] b. Add each reaction component in sequence in the sterilized PCR tube:

[0051] 5 × TTBL 4μl

[0052] DNA 1ng

[0053] TTE Mix V1 5μl

[0054] wxya 2 O make up to 20 μl

[0055] c. Use a pipette to gently pipette 20 times to mix thoroughly.

[0056] d. Place the PCR tube in the PCR instrument and set the following reaction program:

[0057] 55°C for 10 minutes; keep warm at 10°C.

[0058] e. Immediately add 5 μl 5×TS to the reaction product, and mix well by gently blowing with a pipette. Place at room temperature ...

Embodiment 2

[0080] 1. Microplate preparation

[0081] According to the experimental scale (10,000 human 293T cells and 10,000 mouse 3T3 cells each), design the size of the microwell plate (the size of the well plate is 1cm×1cm), and etch microwells on the silicon wafer as the initial mold, the depth of the microwells is 30μm and the microwell The hole size is 30 μm, and the hole spacing is 30 μm.

[0082] Next, polydimethylsiloxane (PDMS) is poured on the silicon wafer. After molding, the PDMS is taken off to become the second mold with micropillars on the plate. The microwell plate used in the final experiment is agar with a concentration of 5%. Sugar (prepared with enzyme-free water), poured on the PDMS micro-column plate to condense and form after hot-melting, and the agarose plate at this time is a micro-well plate with a certain thickness after being peeled off ( figure 1 ). When saving, add DPBS-EDTA mixture that is harmless to cells, cover and store in a 4-degree refrigerator, re...

Embodiment 3

[0116] Put 50,000 CD34 + Cells were added to a 3cm×3cm microwell plate to reach a drop-out rate of about 10%, and excess cells were washed away. Add the molecularly labeled magnetic beads into the microwell plate to achieve a hole drop rate of over 99%, and wash away the excess microwell labeled magnetic beads.

[0117] All the other steps are the same as in Example 2.

[0118] After the gene sequencing library was constructed, it was sent to Novogene for sequencing, using the HiSeq 4000PE125 sequencing strategy. Construction of quadruple CD34 using Microwell-seq high-throughput sequencing platform + The library is used as a duplicate, and the gene expression profile is obtained after splitting, screening and comparing. By importing this matrix file into R language analysis, the matrix data can be converted into a visualized graph. Figure 7 CD34 + Cellular gene expression difference heat map, it can be known that human CD34 + Cells can be divided into 7 subpopulations a...

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Abstract

The invention discloses a molecular marker micro-bead and a high-throughput single cell sequencing method based on the same. The molecular marker micro-bead for high-throughput single cell sequencing comprises a micro-bead body and a link-coupled oligonucleotide chain serving as a molecular marker, the oligonucleotide chain comprises a universal primer sequence, a poly T tail, a molecular tag sequence and a cell tag sequence, wherein the universal primer sequence serves as a primer binding area in PCR (polymerase chain reaction) amplification, the poly T tail is combined with a poly A sequence of mRNA, the molecular tag sequence marks combined mRNA, and the cell tag sequence marks cells derived from the mRNA. The molecular marker sequence of the molecular marker micro-bead is divided into four functional areas including the universal primer sequence, the cell tag sequence, the molecular tag sequence and the poly T tail respectively. The high-throughput single cell sequencing method based on the molecular marker micro-bead can acquire specific transcriptome information of tens of thousands of single cells once, and a complete experiment is low in cost, short in time and high in throughput and has an excellent application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a molecular marker microbead and a high-throughput single-cell sequencing method based on the molecular marker microbead. Background technique [0002] Traditional flow cytometry analysis can quickly perform multi-parameter quantitative analysis on thousands of cells. Fluorescent flow sorting is to pretreat a large number of cells that need to be sorted by surface antigens or fluorescent markers, and can flexibly sort the required cell subpopulations according to the required parameters. The modified flow cytometry fluorescent sorting system can also complete single-cell analysis if the cell density dilution is controlled. However, the cost of various types of antigens is relatively high for flow cytometry instruments. Although the functions are powerful, they mainly perform quantitative grouping analysis of a large number of cells. [0003] For some precious samples, it is difficu...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/02
CPCC12Q1/6806C12Q1/6869C12Q2535/122C12Q2563/149C12Q2563/185C12Q2525/173C12Q2531/113
Inventor 郭国骥赖淑静叶昉
Owner ZHEJIANG UNIV
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