KR20230019056A - single cell RNA-seq analysis in continuous medium without compartmentalization - Google Patents

Abstract

The present invention relates to a method for analyzing single cell transcripts in a continuous medium without intercellular compartmentalization, which can capture the single-cell transcripts at one location without complicated devices, and which greatly simplifies a complex and laborious single-cell RNA sequencing process. The present invention has similar performance to existing single cell analysis methods and can be widely applicable in areas where single cell RNA sequencing is utilized.

Description

Single cell transcriptome analysis method in continuous medium without compartmentalization between cells {single cell RNA-seq analysis in continuous medium without compartmentalization}

The present invention relates to a method for analyzing single cell transcripts in a continuous medium without intercellular compartmentalization.

Single-cell RNA sequencing (scRNA-seq) has been a major means of identifying biological heterogeneity and has been recognized over the past decade as a major criterion for defining distinct cellular states, types and phenotypes. Transcriptional profiling of individual cells provides information about changes in the functional status of heterogeneous populations. The versatility of scRNA-seq has been exploited in a new strategy to easily obtain large numbers of single-cell transcripts. However, despite the development of large-scale parallel approaches, the prior art requires large sample sizes, cumbersome experimental methods, and expensive equipment.

Compartmentalization of single cells is essential to capture single cell transcriptomes, as each cell must be isolated from each other and placed in separate reaction chambers to block interactions with the outside world to be individually barcoded for subsequent cell lysis. Conventional methods such as manual cell division or fluorescence-activated cell sorting (FACS) allocate one well for each cell as a reaction zone, providing scalability for parallel profiling of single cells. (scalability). Recently developed massively parallel approaches also involve compartmentalization of single cells. While microwell-based technology distinguishes physical cells from single cells, droplet microfluidics separates them into droplets. Another technique, combinatorial indexing, utilizes split-pool barcoding of nucleic acids for individual labeling of single cells, which requires separation of cells in each well.

All methods that rely on spatial segmentation are cumbersome and expensive. Single-cell transcriptome capture technology, which does not require physical compartmentalization of cells, has advantages over existing single-cell analysis methods. Because the experiment is performed in a continuous medium, there are virtually no restrictions on scaling up the experiment, enabling massively parallel single-cell RNA sequencing at a slight increase in cost. In addition, since a portion of the sample is inevitably lost when cells are compartmentalized, more samples can be secured. Importantly, since no complicated equipment is required for cell compartmentalization, scRNA-seq can be used in more laboratories. Despite these many advantages, the capture of single-cell transcripts without spatial separation of cells has been difficult because it is difficult to eliminate intercellular mRNA contamination by molecular diffusion.

Accordingly, the present inventors developed Onepot-Seq, a new method for capturing single cell transcripts without physical compartmentalization such as droplets or microwells. Onepot-Seq is space-time limited for each cell, and minimal diffusion of cell contents is formed around the cell after gentle lysis. A sparse distribution of both beads and cells is characterized by the inclusion of beads that specifically capture transcripts of near-single cells in a continuous fluid medium.

Republic of Korea Patent Publication No. 10-2019-0069456

One aspect of the present invention is 1) immobilizing cells and beads in a buffer solution; 2) adding a dissolution solution so that a dissolution solution having a lower density than the buffer solution is positioned on the buffer solution; and 3) capturing the intracellular transcripts dissolved by the lysis solution to the beads.

One aspect of the present invention is 1) immobilizing cells and beads in a buffer solution; 2) adding a dissolution solution so that a dissolution solution having a lower density than the buffer solution is positioned on the buffer solution; and 3) capturing the intracellular transcripts dissolved by the lysis solution on the beads.

Step 1) is a step of immobilizing cells and beads in a buffer solution, and step 1) may be adding cells to beads located in a buffer solution.

The cell is a target cell for analysis and refers to all types of cells having transcripts.

The bead is configured to capture intracellular transcripts dissolved in step 3) described later, and the size, material, and shape can be arbitrarily selected as long as the present invention functions to capture the desired intracellular transcripts, and It may be marked with a barcode for the convenience of classification of the corpse.

In one embodiment of the present invention, the bead is located in the effective radius (Re) of the cell, and the effective radius is an effective distance for the bead to capture the transcript of the cell, and the effective radius is calculated as follows It can be.

(Where λ is the number of beads in the reaction area and ρ is the density of beads in the 2D surface)

If the bead is not located within the effective radius (Re) of the cell, it is difficult to obtain the effect of the present invention because it is difficult for the transcript of the cell to be captured by the bead.

The buffer solution is a solution in which the above-described cells and beads are immobilized, and as will be described later, may be made of a component that has a higher density than the lysis solution capable of dissolving cells and allows the lysis solution to diffuse, and the components of the buffer solution are As long as it performs the function of a buffer solution required in the present invention, it may be arbitrarily selected, and specifically, it may be a mixture of 6% Ficoll PM-400, 20mM EDTA, and 0.2M Tris pH 7.5.

Step 2) is a step of adding a dissolution solution so that a dissolution solution having a lower density than the buffer solution is located on the buffer solution, and if the dissolution solution has a lower density than the buffer solution, a known material may be used. A 20% N-lauroylsarcosine sodium solution and the aforementioned buffer solution (6% Ficoll PM-400, 20mM EDTA, 0.2M Tris pH 7.5) may be mixed at a ratio of 1:1.

As will be described later, the dissolution solution may be positioned on the buffer solution to diffuse in the direction of the buffer solution, that is, the dissolution solution may not be mixed with the buffer solution. To this end, when the dissolution solution is added, the edge of the pipette tip may be placed in contact with the buffer solution and then slowly injected so that the dissolution solution is evenly spread. The addition of such a dissolution solution can be performed by using a dissolution solution having a lower density than the buffer solution, and the dissolution solution is added so that it is located on the buffer solution, but is not added so as not to mix with each other, so that the dissolution solution is positioned to be distinguished from each other. It may diffuse in the direction of the buffer solution to lyse the cells. That is, in order to minimize the movement of beads and cells in the buffer solution, a lysis solution having a lower density than the buffer solution is slowly piled up, and then the cells are lysed through diffusion of the lysis solution. In the conventional single cell analysis experiment technology, there was a difficulty in performing intercellular space separation with separate equipment to prevent the problem of capturing mRNAs from multiple cells in one bead. Through the lysis method, single cell analysis can be performed without spatial separation using separate equipment and without mixing between cells.

Step 3) is a step in which the intracellular transcripts dissolved by the lysis solution are captured by the beads. As described above, the lysis solution is not directly mixed with the buffer solution, but is located on the buffer solution so that the lysis solution becomes a buffer. Cells may be lysed while diffusing in the direction of the solution.

The dissolution may be performed for 5 to 30 minutes, 5 to 25 minutes, 7 to 23 minutes, 10 to 20 minutes, 13 to 17 minutes, 14 to 16 minutes, or 15 minutes after the addition of the dissolution solution in step 2). If the time range is shorter than the above range, cell lysis and/or intracellular transcripts may not be sufficiently captured by the beads, and if the time range is longer, transcripts derived from two or more cells are captured and analyzed by one bead due to diffusion of the transcripts. accuracy may decrease.

In the present invention, in the single-cell transcriptome analysis method, the ratio of beads: cells may be added at a number ratio of 100: 1 to 100: 10, which greatly increases the total volume, making analysis difficult or low efficiency Losing can be prevented.

In the present invention, the single cell transcriptome analysis method includes mixing by shaking the plate up and down and left and right to prevent cells and beads from aggregating in the well, and leaving the cells and beads to settle to the bottom of the well Further steps may be included.

In the present invention, the single-cell transcriptome analysis method may further include, in the step of adding the lysis buffer, slowly adding the lysis buffer by aligning the edge of the pipette tip in a straight line with the water surface and moving in a circular motion so that the added flow rate spreads as evenly as possible. can

In the present invention, the single cell transcriptome analysis method may further include, after the step of adding the solution, culturing the plate to wait for the cells to be lysed and the mRNA to be captured by the beads. In this case, due to the difference in density, the lysis solution may be positioned on an upper layer of the buffer solution containing the cell beads, and the lysis solution may further include spreading toward the bottom of the well.

In the present invention, the single cell transcriptome analysis method may be that cells are completely lysed within 5 to 15 minutes after adding the lysis buffer.

In the present invention, the single cell transcriptome analysis method comprises the steps of adding 6X SSC to beads after the bead capture step; After centrifugation of the beads, washing twice with 6X SSC and washing with Maxima 5X RT buffer may be further included.

The method of the present invention can capture single cell transcripts at one location without complicated devices, and the complicated and laborious single cell RNA sequencing process is greatly simplified. The present invention has similar performance to existing single cell analysis methods and is widely applicable in areas where single cell RNA sequencing is utilized. In addition, since the analysis is performed in one connected space without distinction between cells, there is no limit to expanding the scale of the experiment.

1 and 2 are drawings and photographs showing the concept of the present invention, Figure 1 is a photograph showing the positional relationship between cells (10um ~ 20um) and micro-sized spherical particles (20um ~ 40um), Figure 2 is It is a diagram showing the cell lysis method.
3 and 4 are experimental results confirming the applicability of the present invention. FIG. 3 is an experimental result confirming the mixing of transcripts by the analysis of the present invention after cell mixing, and FIG. 4 is a transcript compared to the conventionally used Dropseq method. This is the result of comparing the capture efficiency.
Figures 5 and 6 show the result of confirming the optimal conditions of the present invention, Figure 5 confirms the efficiency according to the cell lysis time, and Figure 6 shows the multiplet formation rate according to the cell density condition, to avoid multiplet formation above a certain level. Cell density conditions were confirmed.
7 to 10 show the results of confirming the effectiveness of the present invention. FIG. 7 is the result of an AMG510 drug perturbation experiment, FIG. 8 confirms whether clusters are formed for each cell line using the tSNE method, and FIG. 9 is a gene for each cell using the present invention. This is the result of confirming the expression level by heatmap, and FIG. 10 shows the results of the experiment by applying the present invention to GSEA and pseudotime analysis.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more specific examples, and the scope of the present invention is not limited to these examples.

Example 1: Onepot-seq analysis of the present invention

Example 1-1. Preparation of cell lines and cell culture

All cell lines except for DC2.4 were obtained from the Korean Cell Line Bank (KCLB) and used. All cell lines were maintained at 37°C and 5% CO ₂ conditions. Human embryonic kidney cells HEK293T and mouse embryonic fibroblasts NIH3T3 were supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin (Thermo Fisher Scientific, USA). cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco, USA). The human acute T-cell leukemia cell line Jurkat, the mouse dendritic cell line DC2.4, the three human adenocarcinomas NCI-H3122 and NCI-H358, and the human colon adenocarcinoma SW480 were cultured in RPI 1640 supplemented with 10% FBS and 1% penicillin-streptomycin.

Example 1-2. mRNA diffusion prediction

Assuming that mRNA diffuses radially symmetrically, the diffusion equation for mRNA after cell lysis is:

where D is the diffusion coefficient of mRNA, c is the concentration of mNRA, R is the distance from the cell, and t is time. Assuming that the cell is a sphere of radius R, and that the concentration of mRNA is the same within the cell, the values for the above equation give the concentration relationship between time and distance:

where C ₀ is the initial concentration of mRNA in the cell and R is the cell radius. The diffusion coefficient of mRNA was assumed to be 1.0 μm2/sec based on the diffusion coefficient of DNA fragments in water or cytoplasm. The cell size and initial mRNA concentration were assumed to be 5 μm and 1.2 μM, and the mRNA concentration in the droplet of Drop-Seq was calculated assuming that the mRNA in the cell was diluted by the volume of the droplet.

Example 1-3. Onepot-seq analysis

Cells were harvested using standard cell culture methods. After removing the medium from the culture flask, the adhered cells were washed once with 1x PBS, and 2ml trypsin (Trypsin-EDTA; Gibco, USA) was injected at 37°C for 3 to 10 minutes, and the trypsins were inactivated in 8ml growth medium. . Cells were washed three times with PBS-BSA (0.1% BSA in 1x PBS) and resuspended in 10ml PBS-BSA to a concentration of 500-1000 cells/μl. Cells are then 40 It was passed through a μm filter and counted with a hemocytometer. Barcode beads used for Onepot-Seq were purchased from Chemgene (cat. No. MACOSKO-2011-10 (V+)). Beads used in Onepot-Seq were washed three times with PBS-BSA, and incubation buffer (6% Ficoll PM-400 (GE healthcare), 20 mM EDTA (Biosesang), 200 mM Tris pH 7.5 to 20,000 beads per 100 μl) (Biosesang)).

After preparing the beads and cells, 900 μl incubation buffer was added to a 12-well plate (TPP®, Switzerland), and 100 μl of beads (20,000 beads per 100 μl in incubation buffer) were added to the wells. Then, the prepared cells were added until the desired concentration was reached. In order to avoid a large increase in the total volume, the volume of the cells added was about 1-10 μl. In order to avoid aggregation of cells and beads in a specific position in the well, the plate was shaken up and down and left and right, and left for 15 minutes to allow the cells and beads to settle to the bottom of the well.

Cell lysis was performed by adding 200 μl of lysis buffer A (1:1 mixture of 20% N-lauroylsarcosine sodium solution (Sigma) and incubation buffer) to the wells to perform diffusion lysis. When adding the dissolution buffer, adjust the edge of the pipette tip in a straight line with the water surface to minimize the flow rate, and then slowly add it while moving in a circular motion to spread it evenly. The plates were then incubated for 15 minutes to allow cell lysis and mRNA capture by the beads. Since the incubation buffer is denser than lysis buffer A, lysis buffer A is placed on top of the cell bead solution and diffuses towards the bottom of the well. In general, cells began to be lysed 5 minutes after the addition of the lysis buffer, most cells were lysed after 10 minutes, and completely lysed after 15 minutes.

After dissolution, 1 ml of 6X SSC (Biosesang, Korea) was added to raise the beads, and 2 ml of the solution was immediately transferred to a cooled 30 ml of 6X SSC in a Falcon tube. To maximize bead recovery, the remaining beads were added to the Falcon tube after adding an additional 1 ml of 6X SSC and pipetting. The beads were centrifuged at 1000xG for 1 minute, washed twice with 1 ml of 6X SSC, and washed with 50 μl of Maxima 5X RT buffer (Thermo Fisher Scientific, USA). Reverse transcription, exonuclease treatment and cDNA amplification were performed as described in the instructions for Drop-Seq. Briefly, the beads were washed once with TE-SDS (TE buffer (Biosesang)), washed twice with TE-TW (TE buffer, supplemented with 0.01% of Tween-20 (Sigma)), and reverse transcription reaction (30 min, 25 °C and 90 min, 42 °C) once with 10 mM Tris pH 8.0 (Biosesang), and the beads were treated once with TE-SDS after exonuclease reaction (45 min, 37 °C). , Washed twice with TE-TW and nuclease-free water (Invitrogen), and then added RT mix for reverse transcription, 30 minutes at room temperature, and 90 minutes at 42 ° C with a rotator. Then, the beads were washed once with 1ml of TE-SDS and twice with 1ml of TE-TW, and the exonuclease treatment was performed by washing the beads with 1ml of 10mM Tris pH8.0, and exonuclease mix (Exonuclease mix) was added and incubated at 37°C for 45 minutes with a stirrer, and the beads were washed once with 1ml of TE-SDS and twice with 1ml of TE-TW. cDNA was divided into 8 tubes by individual Onepot-seq well reaction, and amplified using KAPA HiFi HotStart PCR Kit (Kapa Biosystems Inc., Switzerland). cDNA amplification was performed with 50 μl PCR solution filled with 4 μl of 10 μM SMART PCR primer (5′-AAGCAGTGGTATCAACGCAGAGT-3′) (IDT), 25 μl of KAPA HiFi DNA polymerase, and 21 μl of nuclease-free water It became. PCR products were pooled and purified using 0.6x AMPure (Beckman Coulter, USA) beads according to the manufacturer's instructions. After the cDNA product was fragmented, it was further amplified using the Nextera XT DNA Library Preparation Kit (Illumina, USA).

As a result, as shown in FIGS. 1 and 2, in the onepot-seq of the present invention, cells and beads are pipetted to a specific surface area (eg, a single well) so that cells and beads are adjacent to each other at a specific location. The distribution of cells and beads can be easily controlled by adjusting the concentration of the solution. And, without disturbing their position, the cells were lysed by diffusion of the lysate. After lysis the beads were recovered and subjected to reverse transcription, exonuclease treatment and amplification.

Experimental Example 1: Validation of the analysis method of the present invention

Experimental Example 1-1. Genomic analysis in mixed samples (Fig. 3)

The effectiveness of the Onepot-seq of the present invention was judged by confirming whether the mixed samples of human and mouse could be distinguished.

Specifically, using the method of Example 1, align reads to hg19-mm10-related references using a STAR aligner, remove barcodes, and separate human and mouse alignment reads from digital expression matrices of human and mouse cells, respectively got it The number of human and mouse transcripts was calculated for each cell barcode, and a barcode with more than 500 transcripts was defined as a cell. The ratio of human transcripts within the cell's barcodes was calculated, and cell barcodes with a ratio of human transcripts greater than 0.9 were defined as human cells, less than 0.1 as mouse cells, and the rest as mixed. The greater of the ratio of the human transcript in the cell and the ratio of the mouse transcript was determined as the species purity of the cell. The multiplet rate was calculated as the ratio of mixed species in total cells, and the cell yield was calculated as the ratio of the number of cells finally obtained through sequencing to the number of initial cells.

As a result, as confirmed in FIG. 3, it was confirmed that high-quality single cell analysis was possible with 137 human cells, 205 mouse cells, and a multiplex ratio of 2.0% in 1000 human-mouse mixed cell experiments performed in a 12-well plate. did

Experimental Example 1-2. Comparison with Drop-seq (Fig. 4)

For Drop-seq, DC2.4 cells and Jurkat cells were seeded in a T75 flask at a density of 1.0x10 ⁶ cells/flask. At 3 days after culture, DC2.4 cells and Jurkat cells were collected. Cells were washed three times with PBS-BSA, passed through a 40 μm filter, and counted. Cells were diluted to 100 cells/μl with PBS-BSA, and Drop-Seq was performed at a bead concentration of 1.0x10 ⁶ cells/ml. To compare Onepot-seq and Drop-seq of the present invention, HEK293T and NIH3T3 cells were used in a 12-well plate at 1000 cells per well. Only mouse cells were used due to size differences between human cells.

Subsequent lysis, sequencing, etc. were performed as described above. After alignment of the transcripts, bam files were randomly sampled for aligned reads at intervals of 10%. At each sequencing depth, we obtained the average original sequencing depth and average UMI of more than 500 transcripts and cell barcodes from the original data.

Assuming that the average UMI saturates with sequence depth, the equation below was used to fit and predict the saturation levels of genes and UMIs:

where x is sequence depth, y is the average number of UMIs or genes, and a, b, and c are fitting parameters.

As a result, as confirmed in FIG. 4, the saturation analysis of UMI using sub-sampling data confirms that the onepot-seq and the drop-seq of the present invention are similar, indicating that the analysis method of the present invention has a similar level of analytical power to the conventional method. Able to know. Specifically, in the case of FIG. 4A, the number of transcripts per cell was captured at a similar level in Drop-seq and Onepot-seq, and as shown in FIG. It can be seen that they have a similar level of analytical power without

Experimental Example 2: Onepot-Seq optimization (lysis time and cell density) (Figs. 5 and 6)

In order to optimize the analysis method of the present invention, optimization conditions for cell lysis time and cell density were confirmed.

Specifically, the same amount of HEK293T and NIH3T3 cells used in the above-described human and mouse mixed cell experiment were prepared. In order to confirm cell lysis conditions, experiments were conducted with different lysis times (100 seconds, 5 minutes, 15 minutes, 30 minutes, 60 minutes). In order to confirm the cell density state, experiments were performed with different cell numbers (1,000, 2,000, 4,000, 8,000, 12,000, 20,000) in 12 wells.

As a result, as confirmed in FIG. 5, it was confirmed that the yield of the cell lysate increased to a saturation level until 15 minutes after dissolution (900 seconds). The cumulative curves of lysis times up to 100 seconds and 5 minutes showed relatively low cell yields and high amounts of unencapsulated transcripts, indicating a low level of analysis. At 30 min after lysis, the cell yield slightly increased, while the purity of the species began to decrease due to cross-contamination from the surrounding environment. Therefore, it can be seen that it is preferable to dissolve for about 15 minutes in order to maximize cell yield and assay level.

Also, as confirmed in FIG. 6 , the multiplet ratio in which two or more cells enter the bead's response range can be expressed as a function of the two-dimensional plane of cell density and the effective capture radius of the response range. In the case of the present invention, it was confirmed that it follows the curve of r = 60 μm, and specifically, it can be seen that the ratio of multiplets increases according to the cell density.

Experimental Example 3: AMG510 (KRAS G12C inhibitor) drug perturbation test (FIGS. 7 to 10)

For single cell expression analysis of cells disrupted by KRAS (G12C) inhibitors, NCI-H3122, NCI-H358, and SW480 with KRAS genotypes of WT, G12C, and G12V, respectively, were cultured for 24 hours and then trypsinized. Then, the cells were washed with PBS and seeded in 12-well plates with wells coated with poly-D-lysine at a density of 1000 per cell in 1 ml of medium. Four wells per cell line were seeded according to different drug concentrations, the plate was shaken to evenly distribute the cells, and then incubated at 37° C. for 8 hours to adhere. After culturing, 700 μl of the medium was removed while maintaining the cells attached to the bottom, and 700 μl of the medium containing various concentrations of AMG-510 (Selleckchem, Cat No. S8830) was added. After drug treatment at 37° C. for 6 hours, 700 μl of medium was removed and 700 μl incubation buffer containing 20000 beads was added to the wells. The beads were incubated for 15 minutes to spread evenly by shaking the plate and to settle. Cells were treated by the onepot-seq method described above, such as being lysed with a lysing solution (FIG. 7).

In the case of cells treated with a KRAS (G12C) inhibitor, a total of 3949 single cells in which at least 1000 genes were detected were identified following sequence alignment and quality filtering using Seurat. After normalizing and scaling the expression matrix, PCA was run using the expression matrix with 2000 variable genes to reduce the dimensionality of the data set.

Then, the first 11 PCA components were selected and t-distributed stochastic neighbor embedding (t-SNE) was performed. Three clusters were identified and each cluster was assigned to each input cell line based on sample features, cell line specific expression markers and SNPs. Expression markers for each cell line were identified with Seurat's FindAllMarkers function with min.pct=0.25 and logfc.threschold=0.25. For the expression heatmap, the scaled single cell expression of the top 10 markers for each cell line was used. Classification of cell cycle stages was performed with Seurat's CellCycleScoring. Each single cell was classified into G2-M, S or G1 phase using cell cycle markers.

As a result, as confirmed in FIG. 8, each cell line corresponds to a unique cluster, whereas the pooled sample consisted of three clusters (Fig. 8 left). Each cell line showed a unique distribution for the four AMG-510 throughputs within each cluster, and for H358, which was inhibited by AMG510, 0uM (drug X) and 0.1/1/10uM (drug O) were differentiated, and drug It was confirmed that clusters were formed by concentration (right side of FIG. 8).

In addition, as confirmed in FIG. 9 , each cell line showed a unique expression pattern with its own label regardless of the amount of AMG-510. Notably, single cells from pooled samples showed a unique expression signature for each cell line and showed preservation of single cell identity during pooling.

Experimental Example 4: Experimental results by applying the present invention to GSEA and pseudotime analysis (FIG. 10)

Experimental Example 4-1. Gene set enrichment analysis

The R package of "msigdbr" was used to analyze the gene set abundance of the transcriptional response signature.

Fisher's measure was used to determine set overlap between each gene set and the top 50 upregulated and downregulated genes based on absolute fold change with adjusted P<0.05 and log fold change >0.5. The P-value was obtained using a likelihood-ratio test, and the gene set used for the analysis was a combination of the “Hallmark” and “Canonical” gene set collections of MSigDB v7.2. AMG-510 per cell line. The results of gene set enrichment analysis of treatment were obtained.

Experimental Example 4-2. Pseudotime analysis

For pseudotime analysis of KRAS (G12C) treatment in H358 cell line, the R package "Monocle3" was applied. Single cell data for the H358 cell line were extracted from the results of KRAS (G12C) inhibitor treatment experiments using Seurat. Data were normalized and pre-processed prior to further analysis. To eliminate cell cycle-dependent effects on cell trajectories, groups of cells with different cell cycle states were aligned using the align_cds function. We reduced the order and cell population with the UMAP algorithm. After fitting a principal graph to the data using the learn_graph function, the node closest to the untreated cell was defined as the origin, the cells were aligned along the trajectory, and the pseudotime was analyzed for each single cell. Moran's I test, which measures multidirectional and multidimensional spatial autocorrelation, was performed to identify differentially expressed genes across single cell trajectories. Genes with q values less than 0.01 were selected as DEGs.

Experimental Example 4-3. Experiment result

As a result of gene abundance analysis of AMG-510 induced genes in each cell line, as shown in FIG. 10A , it was confirmed that KRAS and related pathways were reduced.

And, as shown in FIG. 10B, in order to capture the continuously changing gene expression patterns in the KRAS (G12C) cell line, similar time analysis was performed by defining the node closest to the untreated cell as the origin, and the cells were aligned along the trajectory.

And, as shown in FIG. 10C, the similar time of single cells showed a binary pattern depending on the treatment rather than continuous at the four doses.

However, as shown in Fig. 10D, various modules were consistently identified, suggesting a heterogeneous drug response within each treated sample. Genes regulated by KRAS, such as PLAU, DCBLD2, and ITGA2, were downregulated over time.

These results show that the Onepot-seq of the present invention can capture drug-induced responses and responses influenced by basic pathways in gene expression.

So far, the present invention has been looked at with respect to its preferred embodiments. Those skilled in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent scope will be construed as being included in the present invention.

Claims (7)

1) immobilizing cells and beads in a buffer solution;
2) adding a dissolution solution so that a dissolution solution having a lower density than the buffer solution is positioned on the buffer solution; and
3) Capturing the intracellular transcript dissolved by the lysis solution to the bead
A single cell transcriptome analysis method comprising a.
According to claim 1,
Step 1) is an analysis method in which cells are added to beads located in a buffer solution.
According to claim 1,
The analysis method in which the beads of step 1) are located in the effective radius (Re (effective radius)) of the cell.
According to claim 1.
The analysis method in which the dissolution solution in step 2) is positioned so as not to be mixed with the buffer solution.
According to claim 1,
The analysis method in which the dissolution by the dissolution solution in step 3) is by diffusion of the dissolution solution in the direction of the buffer solution.
According to claim 1,
The analysis method in which the dissolution in step 3) is performed for 5 to 30 minutes after the addition of the dissolution solution.
According to claim 1.
Step 3) is an analysis method in which the intracellular transcripts dissolved by the lysis solution are captured by the beads.

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