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A single-cell, cell-based technology used in molecular biology and drug development to solve problems such as slowness
Pending Publication Date: 2022-04-15
SINGLERON NANJING BIOTECHNOLOGIES LTD
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Problems solved by technology
On the other hand, the slow development of aging phenotypes wins the challenge for improving drug treatment strategies such as drug combinations
Method used
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Embodiment 1
[0077] Drug treatment, scRNA-seq, and sequencing data analysis
[0078] K562 cells were cultured in medium and then exposed to 50 nM oxaliplatin. After 0, 2, 6, and 24 hours of exposure, cells were washed and resuspended in PBS, and samples were denoted as K00_0, K02_50, K06_50, K24_50. Through single cell 3' gel beads, Microfluidic chips and library kits handle single cells. Library sequencing was performed on an Illumina sequencer.
[0079] Compositional changes of cell line populations are visualized by UMAP plots. Cells were subjected to Gene Set Enrichment Analysis (GSEA).
[0080] Total RNA was isolated using the AllPrep DNA / RNA Mini Kit (Qiagen, Cat. No. 80204), and measured using a Qubit2.0 Fluorometer (Life-Technologies, CA, USA) and a NanoPhotometer Spectrophotometer (IMPLEN, CA, USA).
[0081] Prepare a concentration of 1 x 10 5 single cell suspension in PBS (HyClone) per mL. Then load the single cell suspension to the microfluidic device, according to the n...
Embodiment 2
[0089] Drug handling, scRNA-seq, and sequencing data analysis
[0090] K562 cells were cultured in medium and then exposed to different concentrations (0 μM, 5 μM, 20 μM) of lysine-specific histone demethylase 1A (LSD1) inhibitors for 0, 6, 24 hours. Cells were washed and resuspended in PBS, and samples were denoted as LSD1_0_0, LSD1_6_0, LSD1_6_5, LSD1_6_20, LSD1_24_0, LSD1_24_5, and LSD1_24_20. Through single cell 3' gel beads, Microfluidic chips and library kits handle single cells. Library sequencing on an Illumina sequencer
[0091]Compositional changes in cell line populations are visualized by t-SNE plots. GSEA was performed on cells considering different datasets (Gene Ontology database and KEGG signaling pathway database) by cluster profiler. Pseudotime trajectories for each single sample were identified by using Monocle2 under default parameters. Pseudo-time trajectories for all cell lines were analyzed, along with the position of each clustered cell on the tra...
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Abstract
The invention relates to a single cell transcriptome sequencing (scRNA-seq) technology which is applied to primary cells or cell lines for high-throughput drug screening and new application of old drugs. The present invention relates to an analytical method for cell-based drug screening, comprising the steps of: culturing a population of cells in a culture environment; exposing the cell population to a drug substance, wherein a clear time monitoring point and a concentration monitoring point of the drug substance are set; after being exposed in a drug substance, a single cell library is formed; performing single-cell RNA sequencing of the single-cell library on a plurality of genes of the independently sorted cells in the cell library; and performing a bioinformatics analysis to determine differentially expressed genes. Furthermore, the present invention relates to the use of an analytical method for cell-based drug screening in which differentially expressed genes are potential marker genes and are used for screening cancer drugs.
Description
technical field [0001] The invention belongs to the fields of molecular biology and drug development. Specifically, the invention relates to mammalian cell culture, single-cell RNA sequencing (scRNA-seq), and drug screening. Background technique [0002] Drug screening can be derived from transcriptomic (RNA), proteomic, and metabolomic data. Transcriptome signatures are obtained by comparing gene expression profiles of cells or tissues before and after treatment with a drug. Drugs developed using previous batch approaches have encountered problems in that a drug that works for some patients may have little or no effect in others, or a drug may gradually lose efficacy over the course of treatment in some patients despite its Appears to be effective in the initial stages of treatment. The above problems can partly be attributed to different levels of heterogeneity, that is, the expression profiles may differ in different individuals or in different tumors in an individual ...
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