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CN-121992068-A - Application of polyethylene glycol in reducing diffusion of cell content in single cell suspension and improving single cell nucleic acid capturing efficiency

CN121992068ACN 121992068 ACN121992068 ACN 121992068ACN-121992068-A

Abstract

The invention provides an application of polyethylene glycol in reducing cell content diffusion in single cell suspension and improving single cell capturing efficiency. When single-cell nucleic acid is captured by using the single-cell suspension containing polyethylene glycol, the diffusion of single-cell contents such as nucleic acid like genome DNA or transcriptome RNA can be effectively reduced, so that the single-cell contents are more aggregated, and the cell flux and the capturing efficiency are effectively improved.

Inventors

  • LUO HONGYU
  • ZHOU XIAOXI
  • HOU HUAN
  • ZHANG ZHE
  • LIU QIAN
  • LIAO SHA
  • CHEN AO

Assignees

  • 深圳华大三箭齐发科技有限责任公司

Dates

Publication Date
20260508
Application Date
20241031

Claims (12)

  1. 1. Use of polyethylene glycol to reduce diffusion of cell contents in single cell suspensions.
  2. 2. Use of polyethylene glycol to increase single cell capture efficiency.
  3. 3. Use according to claim 1 or 2, wherein the polyethylene glycol has an average molecular weight of 400-6000, preferably 2000.
  4. 4. Use according to any one of claims 1 to 3, comprising mixing polyethylene glycol, cells with a buffer system to prepare a single cell suspension.
  5. 5. Use according to claim 4, wherein the concentration of polyethylene glycol in the single cell suspension is 0.4% -2%, preferably 2%.
  6. 6. The use according to claim 4 or 5, wherein the buffer system has a pH of 7.0-7.6, preferably 7.2-7.6, preferably wherein the buffer system is a phosphate buffer system or a citrate buffer system.
  7. 7. The use according to any one of claims 4-6, wherein the single cell suspension further comprises bovine serum albumin, preferably the concentration of bovine serum albumin in the single cell suspension is 0.01w/v% to 1w/v%, preferably 0.04w/v% to 1w/v%, most preferably 0.04w/v%.
  8. 8. A kit comprising polyethylene glycol, a buffer system, a spatial chip or microbead for capturing nucleic acids in cells, and optionally bovine serum albumin.
  9. 9. The kit of claim 8, wherein probes are attached to the space chip or the surface of the microbead, the probes sequentially comprising a barcode sequence and a capture sequence from 5 'to 3', the barcode sequences attached to the probes at different positions on the space chip being different from each other, the barcode sequences attached to the probes on different microbeads being different from each other, preferably the probes further comprising a unique molecular tag, The unique molecular tag is located 5' to the capture sequence, preferably between the barcode sequence and the capture sequence.
  10. 10. A method for capturing nucleic acid in a cell, the method comprising: (1) Providing a single cell suspension comprising polyethylene glycol, a buffer system, and optionally bovine serum albumin; (2) Contacting the single cell suspension prepared in step (1) with a spatial chip or microbead for capturing nucleic acid in the cells to capture nucleic acid in the cells.
  11. 11. The method according to claim 10, wherein probes are attached to the surface of the spatial chip or microbead, said probes comprising in sequence from 5' to 3' a barcode sequence and a capture sequence, the barcode sequences attached to the probes at different positions on the spatial chip being different from each other, the barcode sequences attached to the probes on different microbeads being different from each other, preferably the probes further comprising a unique molecular tag located in the 5' direction of the capture sequence, preferably between the barcode sequences and the capture sequence.
  12. 12. The method according to claim 10 or 11, wherein the density of cells on the spatial chip is at least 2 x 10 4 cells/cm 2 , such as at least 4 x 10 4 cells/cm 2 , at least 6 x 10 4 cells/cm 2 , or at least 7 x 10 4 cells/cm 2 , preferably the density of cells on the spatial chip is 2 x 10 4 -6×10 4 cells/cm 2 .

Description

Application of polyethylene glycol in reducing diffusion of cell content in single cell suspension and improving single cell nucleic acid capturing efficiency Technical Field The invention belongs to the field of gene sequencing, and particularly relates to application of polyethylene glycol in reducing diffusion of cell contents in single-cell suspension and improving single-cell nucleic acid capturing efficiency. Background With the rapid development of single cell sequencing technology, single cell sequencing has increased in demand for cell flux, and some large research projects may cover millions of cells. The greater the number of cells in a single cell study, the greater the number of captured cell types and the more accurate the differences between the cell subsets studied. The high-throughput single-cell sequencing can accurately analyze the composition information of the sample cells by virtue of the extremely high resolution, and the high-throughput sequencing mode is combined, so that the gene structure and the gene expression state of the single cells are revealed on a large scale, and the heterogeneity among the cells is reflected. However, as single cell throughput requirements increase, different single cell sequencing techniques encounter certain problems. For example, single cell RNA capture diffusion increases in single cell RNA capture based on planar capture after flux enhancement, transcriptomes cannot be separated between cells, and higher double cell rate/multicellular rate occurs, resulting in failure to accurately analyze transcriptomes of single cells. Single cell capture techniques require resuscitating solid tissue or frozen cells to prepare a single cell suspension, which is mainly resuspended in PBS containing a percentage of Bovine Serum Albumin (BSA) or with medium. Counting the resuspended cells, and dripping a certain volume of cell suspension onto the chip after counting. When the input amount of cells on a 1cm x 1cm chip is 2 ten thousand or less, the cell capturing efficiency is over 70 percent, but when the input amount of cells on a 1cm x 1cm chip is increased to 4 ten thousand or even 6 ten thousand, RNA of each captured cell is seriously diffused by single cell detection, the cell outline is blurred, a single cell transcriptome cannot be accurately circled, and the cell capturing efficiency is affected. Thus, there is a need in the art to develop a method of reducing diffusion of cell content in single cell suspensions and/or improving single cell nucleic acid capture efficiency. Disclosure of Invention In view of the problems in the prior art, that is, the problem that the cell capturing efficiency is reduced when the input amount of cells is excessive in single cell (planar) capturing, the present invention surprisingly found that the diffusion of the single cell content can be reduced when single cell sequencing is performed by re-suspending cells using a solution containing polyethylene glycol (PEG), thereby effectively improving the cell capturing efficiency and throughput. In a first aspect, the invention provides the use of polyethylene glycol to reduce diffusion of cell content in a single cell suspension. In a second aspect, the invention provides the use of polyethylene glycol to increase the efficiency of single cell capture. In a third aspect, the invention provides a kit comprising polyethylene glycol, a buffer system, a spatial chip or microbead for capturing nucleic acids in cells, and optionally bovine serum albumin. In a fourth aspect, the invention provides a method for capturing nucleic acid in a cell, the method comprising: (1) Providing a single cell suspension comprising polyethylene glycol, a buffer system, and optionally bovine serum albumin; (2) Contacting the single cell suspension prepared in step (1) with a spatial chip or microbead for capturing nucleic acid in the cells to capture nucleic acid in the cells. The invention captures nucleic acids (e.g., transcriptome) in cells by plating cells in a single cell suspension comprising polyethylene glycol onto a solid phase (i.e., chip) surface with probes, thereby capturing nucleic acids (e.g., transcriptome) in cells using probes on the solid phase surface, or by combining cells in the single cell suspension with microbeads (e.g., magnetic beads) with probes using microfluidic technology, thereby capturing nucleic acids (e.g., transcriptome) in cells using probes on the microbead surface. Experimental results show that the polyethylene glycol can reduce the diffusion of cell contents, so that single cells are more aggregated during planar capturing, and the cell capturing efficiency is improved. In addition, because the single cell suspension containing polyethylene glycol causes the cells to be more concentrated on a plane with a certain area, more cells can be put into the plane with the same area, and the flux of single cell sequencing is further improved. Drawings In order to more clearl