Novel single-celled spatial transcriptome technology for tissue

A space transcription, single-cell technology, applied in the field of single-cell space transcriptome technology

Active Publication Date: 2021-08-31
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although there have been many reports on the use of samples obtained by laser microdissection for transcriptome sequencing of large numbers of cells, these studies often require the collection of tens to thousands of cells

Method used

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  • Novel single-celled spatial transcriptome technology for tissue
  • Novel single-celled spatial transcriptome technology for tissue
  • Novel single-celled spatial transcriptome technology for tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0131] LCM-SCRB-seq method of the present invention

[0132] 1. Preparation of Cell Collection Solution

[0133] The buffer solution (containing Phusion HF buffer diluted 500 times, 2U / μL RNase inhibitor, 0.2% Triton X-100) was prepared according to the following composition.

[0134] Table 1

[0135] Reagent volume Phusion HF buffer (New England Biolabs, USA) 2μL RNase Inhibitor 35.8μL 2% Triton X-100 71.6μL Nuclease-free Water 606.6μL total capacity 716μL

[0136] Add 10 μL of cell collection solution (8 μL of buffer and 2 μL of cell-tagged primer (E3V6NEXT, US14898030A1) at a concentration of 2 μM) to a single nuclease-free PCR tube, or inside the lid of an LCM 96-well collection plate Add 7 μL of cell collection solution (5 μL of buffer and 2 μL of cell-labeled primers at a concentration of 1 μM) to each well, cover the 96-well plate, and place the plate with the cover down and put it in a 4°C refrigerator for later use....

Embodiment 2

[0188] Characteristic analysis of the LCM-SCRB-seq method of the present invention

[0189] 1. Quality detection of cDNA in single cells captured in situ from tissues

[0190] As described in Example 1, the LCM-SCRB-seq method of the present invention was used to sort single cells by laser microdissection, mRNA inversion, cDNA amplification and purification, and the obtained cDNA was analyzed by Agilent 2100 for quality inspection.

[0191] figure 2 The quality inspection results show that the full-length cDNA obtained by the LCM-SCRB-seq method of the present invention accounts for more than 50% of the length of more than 1000bp, and even exceeds about 50% of the single-cell transcriptome gold standard SMART-seq2. Database building requirements.

[0192] 2. Sensitivity detection

[0193] Using the LCM-SCRB-seq method of the present invention (as described in Example 1), the oocytes of 30-day normal mice were constructed and sequenced.

[0194] The results showed that at th...

Embodiment 3

[0199] Single-cell transcriptome sequencing of 30-day mouse oocytes using the LCM-SCRB-seq method of the present invention

[0200] 1. Preparation of Cell Collection Solution

[0201] Add 7 μL of cell collection solution (5 μL of buffer and 2 μL of cell-labeled primers at a concentration of 1 μM) to each well in the cover of the LCM 96-well collection plate. The buffer solution is prepared according to Table 1 in Example 1, and the 96 wells are covered Plates, with the plates covered, placed in a 4°C refrigerator for later use.

[0202] 2. Freezing section and staining

[0203] Take out the 30-day mouse ovarian tissue from -80°C, freeze the section at 20 μm, paste the tissue section on the filmed PEN slide that has been pre-cooled in ice, fix it in 95% ethanol for 2 minutes, and stain with methyl violet staining solution for 1 minute and 50 seconds , after drying with ethanol gradient dehydration, air-dry the slides naturally for a few seconds, and balance the temperature sl...

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Abstract

The invention provides a single-celled spatial transcriptome analysis method. Specifically, the invention provides a single-celled spatial transcriptome capable of detecting an undissociated state. According to the method provided by the invention, single cells can be collected in a high flux manner on the basis of reserving spatial position information of cells in tissue, quantified cellular transcriptome data with high sensitivity and high repeatability are obtained through secondary high-flux sequencing, single-celled spatial transcriptome sequencing data of each target cell are mapped to corresponding spaces of a tissue sample based on three-dimensional coordinate values of each target cell, and thus, a single-celled spatial transcriptome sequencing data set of the tissue sample is obtained. The method provided by the invention can be extensively applied to quantitative research on single-celled gene expression of normal and diseased tissue and provides a more efficient and accurate tool for researching states and functions of cells of the tissue.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, the invention relates to a single-cell spatial transcriptome technology for detecting cells in a non-dissociated state. Background technique [0002] Quantitative measurement and study of gene expression in cells in normal and diseased tissues is critical information for understanding normal tissue function and diseased tissue abnormalities. In recent years, the rapid development and application of single-cell transcriptome technology has broken through the limitation that traditional transcriptome technology can only obtain the average gene expression information of a large number of cells, and provided the ability to analyze the gene expression profile of a single cell, enabling further analysis at single-cell resolution. Various biological problems become possible. A variety of single-cell transcriptome library construction methods emerge in endlessly, greatly improving the through...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869G01N1/30G01N1/28G01N1/42
CPCC12Q1/6869G01N1/30G01N1/286G01N1/42G01N2001/2886G01N2001/302G01N2001/305C12Q2523/319C12Q2535/122C12Q2563/185
Inventor 郭妍胡苗苗邵志峰
Owner SHANGHAI JIAO TONG UNIV
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