Single cell sorting method and application thereof
A single-cell sorting technology, applied in the field of biomedicine, can solve problems such as inability to retain, and achieve the effects of saving reagent consumption, improving the success rate of construction, and requiring high active cell counts
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Embodiment 1
[0054] This embodiment provides a single cell titration sorting system, comprising the following steps:
[0055] 1. Configuration of cell viability detection reagents: Use sterile PBS to configure trypan blue to 100 μM for use;
[0056] 2. Cell culture: add 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin DMEM culture solution to the culture dish and carry out cell culture in a 37°C incubator;
[0057] 3. Sample preparation: wash the tumor cells growing on the culture dish with 37°C pre-warmed PBS for 3 times, and wash with 0.25% TrypLE TM Express trypsin dissociates and digests adherent cells, terminates the digestion with complete culture medium containing 10% FBS, centrifuges at 500 g for 5 minutes at room temperature, discards the supernatant, resuspends the cell pellet with complete culture medium, prepares a suspension, and adjusts the suspension density to 1×10 5 Each cell / mL, take 50ul for use, add 100μM trypan blue solution at a ratio of 1:10,000 for sta...
Embodiment 2
[0064] Based on the method in Example 1, the single cells selected by CellenONE X1 single-cell titration were subjected to full-length transcriptome analysis:
[0065] 1. Add 1.5 μL calcium-magnesium-free PBS solution containing 3U RNase inhibitor to each well of a 96-well plate in advance, use CellenONE X1 to perform single-cell titration sorting, titrate 1 cell per well, and select according to the remaining nozzle area Photos of single cells excluding empty wells, multicellular wells and cells stained with trypan blue;
[0066] 2. Randomly select individual cells from the four wells in step 1 for full-length transcriptome analysis: apply The SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing method performs reverse transcription on single cells in each well and then uses XT DNA Library Preparation Kit and XT DNA Library Preparation Kit method for library construction and quality inspection,
[0067] The result is as Figure 3-6 shown, where:
[0068] image 3 Sche...
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