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Construction method of single-cell or trace sample full-length transcriptome library for nanopore sequencing

A technology for nanopore sequencing and library construction, applied in chemical libraries, biochemical equipment and methods, combinatorial chemistry, etc. Effect

Pending Publication Date: 2021-05-28
BIOMARKER TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

thus ignoring information about the transcript as a whole

Method used

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  • Construction method of single-cell or trace sample full-length transcriptome library for nanopore sequencing
  • Construction method of single-cell or trace sample full-length transcriptome library for nanopore sequencing
  • Construction method of single-cell or trace sample full-length transcriptome library for nanopore sequencing

Examples

Experimental program
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Effect test

Embodiment 1

[0046] This embodiment provides a method for constructing a full-length transcriptome library of a single cell or micro sample for nanopore sequencing (the basic process is as follows: figure 1 shown), including the following steps:

[0047] 1. Single cell lysis

[0048] Prepare 10×Reaction Buffer according to Table 1, add it to a single-cell tube, flick to mix, and centrifuge immediately (the lysis buffer contains detergent, bubbles must be avoided when flicking and mixing), and lyse at room temperature for 5 minutes.

[0049] Table 1 Recipe of single cell lysis reaction buffer

[0050] components Dosage 10× Lysis Buffer 19μL RNase inhibitor 1μL total capacity 20 μL

[0051] 2. Reverse transcription

[0052] (1) Take a 0.2mL centrifuge tube and prepare reverse transcription reagents according to Table 2.

[0053] Table 2 RT Reaction System Recipe (1)

[0054]

[0055]

[0056] (2) Put the sample on ice, and add 2 μL of 3’SMART-S...

Embodiment 2

[0116] This embodiment provides a method for constructing a full-length transcriptome library of a single cell or micro sample for nanopore sequencing (the basic process is as follows: figure 1 shown), the difference from the method in Example 1 is that the end repair reaction condition in step 5 is to first add 2 μL of FFPE Master Mix and 3.5 μL of FFPE buffer to the PCR purified product, react at 20°C for 15min, and then Add 2 μL of Endprep Master Mix, 3.5 μL of Endprep buffer and water to the reaction system, react at 20°C for 30 minutes, and at 65°C for 10 minutes. The linker ligation reaction condition in step 7 is 22°C for 1.5 hours.

experiment example

[0120] The full-length transcriptome library construction method for single-cell or micro-sample for nanopore sequencing in Examples 1 and 2 and the comparative example is used to construct a full-length transcriptome library for a single-cell sample, wherein the library sequencing quality and library sequencing pass the pore The speed test results are as follows image 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 and Figure 8 As shown, the off-machine results of the measured data obtained from the two samples are shown in Table 9, and the statistical results of the full-length sequence of the measured transcripts obtained are shown in Table 10. The results show that the sequencing effects of Example 1 (N01, N02) and Example 2 (N03, N04) are equivalent, and compared with the comparative example (N05, N06), the library quality of Example 1 and 2 is significantly improved ( image 3 , Figure 5 and Figure 7 ), the hole speed is relatively stable in a short time ( ...

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Abstract

The invention relates to the technical field of single cell sequencing, in particular to a single cell or trace sample full-length transcriptome library construction method for nanopore sequencing. The library construction method provided by the invention comprises the following steps: splitting a single cell or a trace sample, reversely recording mRNA by adopting a Smart-seq2 method to prepare cDNA, performing PCR amplification by taking the cDNA as a template, performing FFPE and end repair on a PCR amplification product, and connecting a nanopore sequencing joint. The method can realize real single cell sequencing, has the advantages of high transcript coverage, high resolution and strong flexibility, and can be applied to differential gene expression analysis, transcript structure analysis, full-length isoform identification and the like of single cells.

Description

technical field [0001] The invention relates to the technical field of single-cell sequencing, in particular to a method for constructing a single-cell or micro-sample full-length transcriptome library for nanopore sequencing. Background technique [0002] For multicellular organisms, although they come from a fertilized egg, as the individual develops, the cells gradually differentiate into different subgroups, each of which expresses different genetic information and performs different physiological functions. For the same tissue, there is also heterogeneity between adjacent cells, such as brain tissue and heart tissue. In particular, a tumor is a collection of mutated cells. Genetic analysis (bulk phase sequencing) is performed on tumor tissue and microenvironmental cells in general. Mixed sequencing of different cells is likely to cover up some key information. Single-cell sequencing can perfectly solve this problem, greatly improving the resolution of sequencing analys...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/6869
CPCC12Q1/6869C40B50/06C12Q2525/191C12Q2535/122C12Q2565/631
Inventor 郑洪坤刘敏陈钰萌张梦龙
Owner BIOMARKER TECH
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