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Single cell transcriptome sequencing method based on third-generation sequencing

A single-cell, transcriptome technology, applied in the field of single-cell sequencing, can solve problems such as material inaccessibility

Active Publication Date: 2020-08-18
BIOISLAND LAB
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the TGS sequencing strategy requires large amounts of raw material for library construction, which cannot be obtained directly from individual cells

Method used

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  • Single cell transcriptome sequencing method based on third-generation sequencing
  • Single cell transcriptome sequencing method based on third-generation sequencing
  • Single cell transcriptome sequencing method based on third-generation sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Principle of single-cell transcriptome sequencing

[0058] The flow chart of single-cell transcriptome sequencing based on nanopore single-cell sequencing of the present invention is as follows figure 1 Shown:

[0059] The single-cell lysate is co-incubated with the reverse transcription system, and the reverse transcription primer is combined with the polyA tail of the single-cell mRNA through poly dT, and the barcode sequence is introduced into the single-cell mRNA, and the mRNA is reverse-transcribed into the first strand under the action of reverse transcriptase cDNA, the barcode sequence realizes the specific labeling of the first-strand cDNA; under the action of terminal transferase, the template-switching primer combines with the first-strand cDNA and extends to obtain double-stranded DNA;

[0060] Using the anchor sequence to perform PCR amplification to obtain the amplified product; using magnetic beads to purify the amplified product, removing the a...

Embodiment 2

[0062] Example 2 Single Cell RNA Reverse Transcription

[0063] (1) Single cell lysis

[0064] Isolate single mouse embryonic stem cells (mESCs), transfer each cell to an independent reaction tube, and add a certain amount of single cell lysate to prepare the lysis system shown in Table 1, shake and mix and incubate at 72°C for 3 minutes to make The cells are fully lysed to release RNA, and the samples are immediately transferred to ice;

[0065] Table 1 Single cell lysis system

[0066] Element Volume (μL) 10% Triton X-100 0.095 RNase inhibitor (40U / μL) 0.05 Reverse transcription primer (5μM) 0.3 dNTPs (10mM) 0.5 Enzyme-free water 1.555

[0067] (2) In vitro reverse transcription reaction

[0068] Add the reverse transcription reaction solution shown in Table 2 directly to the single-cell lysed sample obtained in step (1). °C for 10 min to obtain cDNA complementary to the RNA, and the cDNA carries a barcode sequence (Barcode)....

Embodiment 3

[0071] Example 3DNA amplification and amplification product purification

[0072] (1) PCR amplification

[0073] Perform PCR amplification reaction on the cDNA obtained in Example 2. The system is shown in Table 3. The conditions are 95°C pre-denaturation for 3 minutes; 98°C denaturation for 20s, 65°C annealing for 30s, 72°C extension for 5min, 3-6 cycles; 98°C Denaturation at ℃ for 20s, annealing at 67℃ for 15s, extension at 72℃ for 5min, 10-20 cycles;

[0074] Table 3 PCR amplification system

[0075] Element Volume (μL) KAPA HiFi HotStart ReadyMix (2×) 6.25 Anchor primer (10 μM) 0.25 cDNA template 5.4 Enzyme-free water 1

[0076] (2) Product purification

[0077] Add SPRI magnetic beads to purify the amplified product. The magnetic beads are 0.4 times the volume of the reaction solution. During the purification process, the magnetic beads and the product are mixed first, incubated at room temperature for 5 minutes, and then placed ...

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Abstract

The invention provides a single-cell transcriptome sequencing method based on third-generation sequencing, which comprises the following steps: (1) carrying out reverse transcription on single-cell RNA by using a reverse transcription primer to obtain cDNA with a bar code; (2) carrying out PCR (Polymerase Chain Reaction) amplification on the cDNA with the bar code, and mixing obtained PCR amplification products with different single cell sources; or mixing cDNA (complementary deoxyribonucleic acid) with barcodes from different single cell sources and then carrying out PCR (polymerase chain reaction) amplification; the reverse transcription primer sequentially comprises an anchoring sequence, a bar code sequence and poly dT from the 5'end to the 3 'end. According to the invention, a bar code sequence is added into a reverse transcription primer to carry out reverse transcription on single-cell full-length RNA; sequences of different single cell sources are marked, DNA amplification products of different sources are mixed to meet the requirement of a third-generation sequencing platform for the template amount, and accurate sequencing of a full-length transcript is achieved through the third-generation sequencing platform.

Description

technical field [0001] The invention belongs to the technical field of single-cell sequencing, and relates to a single-cell transcriptome sequencing method based on third-generation sequencing. Background technique [0002] The emergence of next-generation sequencing technology (Next-generation sequencing, NGS) has pushed molecular biology research into an era of high-throughput development. Using NGS to generate a large amount of transcriptome data is widely used in basic biological research and medical health. field. Traditional sequencing methods require a large number of starting cells to obtain sufficient sequencing templates, and the obtained data is also the result of mixing all cells, especially in RNA sequencing, where the differences between cells are submerged in the average value. [0003] Single-cell RNA sequencing technology (scRNA-seq) emerged at the historic moment, and in the past decade, single-cell transcriptome sequencing based on NGS platforms has made ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6806C12Q1/6869
CPCC12Q1/6806C12Q1/6869C12Q2521/107C12Q2531/113C12Q2563/185C12Q2525/173C12Q2523/308C12Q2565/631
Inventor 范小英苏丹
Owner BIOISLAND LAB
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