Induced sputum single-cell suspension as well as preparation method and application thereof

Abstract

The invention relates to an induced sputum single-cell suspension as well as a preparation method and application thereof. The preparation method comprises the following steps: obtaining a filtered sputum suspension to be detected; performing low-temperature centrifugation; centrifuging the sample, and removing supernate; precipitating the cells, adding DPBS which does not contain calcium and magnesium ions, lightly mixing with a transfer pipette, and repeatedly inverting the transfer pipette to enable the cell pellets to suspend again; filtering and collecting filtrate; performing low-temperature centrifugation on the filtrate collected in the previous step, and removing supernate without damaging cell precipitation; adding a proper amount of DPBS free of calcium and magnesium ions into the cell precipitate obtained in the previous step, and gently mixing with a pipette; and adjusting the volume of the suspension to obtain the target cell concentration, thereby obtaining the single-cell suspension. According to the present invention, the induced sputum single cell suspension is prepared, such that a large amount of sputum single cells can be enriched, and the obtained induced sputum single cell suspension can be used for single cell transcriptome sequencing so as to well detect the gene expression condition of the sputum cells, such that the airway inflammation pathogenesis can be easily explored, and the clinical treatment can be easily guided.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to induced sputum single cell suspension and a preparation method and application thereof. Background Art

[0002] Induced sputum cytology can be used to reflect the type of airway inflammation. Sputum examination is very valuable for the diagnosis of lung diseases such as bronchial asthma, tuberculosis, bronchitis, paragonimiasis, and lung tumors, and is of great value in guiding the clinical treatment of chronic airway inflammatory diseases. Using induced sputum cytology classification, the efficacy of drugs in clinical work can also be judged. However, the current clinical induced sputum cytology detection is mainly based on the morphology of induced sputum cells, which limits the further understanding of airway inflammatory cells, especially the further study of the reaction mechanism within inflammatory cells.

[0003] Currently, there is no relevant technology developed for the detection of gene expression in induced sputum cells.

[0004] The present invention helps to reveal the gene expression of induced sputum cells by developing a single-cell transcriptome methodology for induced sputum cells, which helps to explore the pathogenesis of airway inflammation and guide clinical treatment. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide an induced sputum single cell suspension and a preparation method and application thereof. The induced sputum single cell suspension prepared by the present invention can be suitable for single cell transcriptome methodological detection.

[0006] The technical solutions for achieving the above-mentioned purpose include the following.

[0007] The first object of the present invention is to provide a method for preparing an induced sputum single cell suspension, comprising the following steps:

[0008] 1) obtaining a filtered sputum suspension to be tested;

[0009] 2) Low temperature centrifugation;

[0010] 3) After centrifugation of the sample, the supernatant was removed without destroying the cell pellet;

[0011] 5) Add DPBS without calcium and magnesium ions to the cell pellet, mix gently with a pipette, and repeatedly invert the tube to resuspend the cell pellet;

[0012] 6) Filter and collect the filtrate in a new centrifuge tube;

[0013] 7) performing low-temperature centrifugation on the filtrate collected in the previous step, and removing the supernatant without destroying the cell pellet;

[0014] 8) Add an appropriate amount of DPBS (Dubler Phosphate Buffered Saline) without calcium and magnesium ions to the cell pellet in the previous step, mix gently with a pipette, and gently pipette 10-15 times or until the cells are completely suspended;

[0015] 9) Adjust the suspension volume to obtain the target cell concentration, that is, a single cell suspension.

[0016] In some embodiments, the parameters of the low-temperature centrifugation are: 1200±10 rpm, 4°C, centrifugation for 4-6 min.

[0017] In some embodiments, the target cell concentration is 700-1200 cells / ul.

[0018] In some embodiments, in step 6), the pore size of the filter used for filtration is 30±5 um.

[0019] In some of the embodiments, in step 6), when using a filter, the filter should be first wetted with 1 ml of DPBS without calcium and magnesium ions. After the cell suspension is filtered, 500-1000 ul of DPBS without calcium and magnesium ions is added to the filter to collect as many filtered cells as possible.

[0020] The second object of the present invention is to provide an induced sputum single cell suspension prepared by any of the above methods.

[0021] The third object of the present invention is to provide an application of any of the above-mentioned methods for preparing induced sputum single-cell suspensions in an induced sputum single-cell transcriptome sequencing method.

[0022] The fourth object of the present invention is to provide a method for single-cell transcriptome sequencing of induced sputum.

[0023] A method for single-cell transcriptome sequencing of induced sputum, comprising the following steps:

[0024] The induced sputum single cell suspension prepared according to any of the above methods,

[0025] Perform scRNA-seq library construction and sequencing.

[0026] In some embodiments, the scRNA-seq library construction and sequencing include the following steps:

[0027] A) GEM generation and labeling;

[0028] Single cell suspension of induced sputum was isolated and labeled by reverse transcription;

[0029] B) Post GEM-RT purification and cDNA amplification;

[0030] C) Library construction and quantification;

[0031] D) Sequencing the constructed library.

[0032] Compared with the prior art, the present invention has the following beneficial effects: the present invention can enrich a large number of sputum single cells by preparing induced sputum single cell suspension, so that the obtained induced sputum single cell suspension can be used for single cell transcriptome sequencing, thereby being able to well detect the gene expression of sputum cells, which is helpful to explore the pathogenesis of airway inflammation and guide clinical treatment, and can also be used for the diagnosis of lung diseases such as bronchial asthma, tuberculosis, bronchitis, paragonimiasis, lung tumors, etc., and has great clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 .Molecular cell atlas of induced sputum, including: A. tSNE diagram of each sample data, one point represents one cell data, and different colors represent different samples; B. tSNE diagram of induced sputum cell data, one point represents one cell data, and different colors represent different cell classifications; C. The proportion of each cell classification in each sample, the ordinate represents the proportion of cells, the ordinate represents samples, and different colors represent different cell classifications; D. tSNE diagram of the expression of a specific gene in a cell, one point represents one cell data, and the color represents the expression intensity of the gene in the cell. The darker the color, the higher the expression of the gene. DETAILED DESCRIPTION

[0034] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0035] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0036] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0037] The present invention is further described in detail below with reference to specific embodiments.

[0038] Example 1

[0039] The method for preparing the induced sputum single cell suspension described in this embodiment comprises the following steps:

[0040] 1) 3% hypertonic saline was used to induce sputum plugs, and 4 times the volume of 0.1% DDT was used to lyse the sputum plugs (water bath, 37°C, 15 min). The sputum suspension was filtered through 200-mesh nylon gauze and placed in a new 5 mL centrifuge tube;

[0041] 2) Use a wide-bore pipette tip to gently and thoroughly mix the cells, take 20 μL of cell suspension, add 20 μL of 0.4% trypan blue, and complete the total cell count and viable cell count assessment within 5 minutes after thorough mixing. Centrifuge the remaining filtered sample at low temperature (1200 rpm, 4°C, centrifuge for 5 minutes);

[0042] 3) During sample centrifugation, a Neubauer blood cell counting chamber was used to assess the total cell count, cell viability, and squamous epithelial cell contamination level;

[0043] 4) After centrifugation of the sample, remove the supernatant without destroying the cell pellet. Store the removed supernatant in another tube, record the volume C ml, and store at -80℃;

[0044] 5) Add 1 ml of DPBS without calcium and magnesium ions to the cell pellet, mix gently with a pipette 5 times, and repeatedly invert the tube to resuspend the cell pellet;

[0045] 6) Use a 30um pore size filter (Miltenyi Biotec, Germany, No. 130-098-458) to filter the sample and use a new 5ml centrifuge tube to collect the filtrate. Note: When using the filter, it should be wetted with 1ml DPBS without calcium and magnesium ions. After the cell suspension is filtered, add 500-1000ul DPBS without calcium and magnesium ions to the cell filter to collect as many filtered cells as possible;

[0046] 7) The collected filtrate in the previous step was subjected to low-temperature centrifugation (1200 rpm, 4°C, centrifugation for 5 min), and the supernatant was removed without destroying the cell pellet;

[0047] 8) Add an appropriate amount of DPBS without calcium and magnesium ions to the cell pellet in the previous step, mix gently with a pipette, and pipette gently 10-15 times or until the cells are completely suspended to make a single cell suspension of 700-1200 cells / ul. Note: According to past experience, the number of cells is lost by half after the whole set of steps. The volume of PBS added in this step can be preliminarily calculated based on the volume of C in step 4;

[0048] 9) Take 20 μL of cell suspension, add 20 μL of 0.4% trypan blue, mix thoroughly and complete the assessment of total cell count and viable cell number within 5 minutes;

[0049] 10) Adjust the suspension volume to obtain the target cell concentration (700-1200 cells / ul).

[0050] 11) Once the target cell concentration is obtained, place the single-cell suspension sample on ice and proceed to the next step of scRNA-seq (single-cell RNA sequencing) within 2 hours.

[0051] Example 2: scRNA-seq library construction and sequencing

[0052] The 10x Genomics single-cell sequencing platform is used. The experimental principle is to generate an "oil-in-water" structure of GEMs (GelBeads-in-emulsion) based on a microfluidic platform. In this "oil-in-water" structure, there is a gel bead with Read1, 10xbarcode, UMI and poly(dT) primer fragments, a target cell, and reagents required for reverse transcription reaction. After GEMs are generated, the gel beads are immediately dissolved to release the primers. At the same time, the cells are also lysed, and the released mRNA is mixed with the reverse transcription reagent and poly(dT), and the full-length cDNA is amplified under appropriate reaction conditions. Then, the cDNA library is constructed by PCR amplification, and the cDNA is fragmented by enzymes. The cDNA library is optimized by fragment size selection. P5, P7, sample index and Read2 are added by PCR to construct the final 3' gene expression library. The gene expression of a single cell can be obtained by high-throughput sequencing. The specific operation steps strictly refer to the official operation instructions provided by 10x Genomics (Single Cell 3' Reagent Kits v3, 10x Genomics, USA).

[0053] The induced sputum single cell suspension obtained in Example 1 was subjected to scRNA-seq and sequencing, comprising the following steps:

[0054] 1) Cell preparation

[0055] Single cell suspensions are quality checked and counted. Generally, the cell survival rate is required to be above 80%. Qualified cells are resuspended to a suitable cell concentration of 700-1200 cells / ul for 10xGenomics Chromium TM System on-machine operation.

[0056] 2) GEM generation and labeling

[0057] GEMs (Gel Bead in Emulsion) were constructed for single cell separation. After GEMs were formed normally, GEMs were collected and reverse transcribed in a PCR instrument to achieve labeling.

[0058] 3) Post GEM-RT purification and cDNA amplification

[0059] GEMs were de-oiled, and the first-strand cDNA was purified and enriched using magnetic beads, followed by cDNA amplification and quality inspection.

[0060] 4) Library construction and quantification

[0061] The cDNA that passed the quality inspection was used to construct the second-generation sequencing library. After fragmentation, ligation of sequencing adapters, sample IndexPCR and other experimental processes, the library was finally quantitatively quality inspected.

[0062] 5) Sequencing

[0063] The constructed library was sequenced using the Illumina NovaSeq platform and the PE150 sequencing mode, measuring 90 to 120G of raw data. The raw image files obtained from the high-throughput sequencing machine were converted into sequenced reads by CASAVA base recognition and stored in FASTQ format.

[0064] scRNA-seq data analysis

[0065] Data quality control

[0066] The raw data was split into different files according to the different Index sequences during library construction, and each file contained Read1 and Read2. FastQC was used to perform a basic assessment of the quality of the raw data; low-quality data was filtered using Trimmomatic software. The filtering criteria were: remove bases with a base quality of less than 3 at the beginning and end of the reads; in the processing process, 4 bases were grouped together, and if the average base quality of the group was less than 15, the base sequence of the group was removed; then FastQC was used to perform a basic assessment of the quality of the clean data after filtering.

[0067] Comparison and quantification

[0068] The software CellRanger (v6.1.1) for analyzing 10X data released on the official website of 10X Genomics was used to perform data quality statistics and compare the data with the human reference genome (GRCh38). The high-throughput single-cell transcriptome results were quantified by identifying the Barcode sequence used to distinguish cells in the sequence and the UMI markers of different mRNA molecules in each cell.

[0069] Based on this method, the single-cell transcriptome of induced sputum cells can be successfully detected, which will be helpful for subsequent scientific research and clinical application development.

[0070] By the method described in Example 1, 10 samples (the clinical information of the subjects is shown in Table 1) were tested according to the above-mentioned scRNA-seq library construction and sequencing method, and the relevant results are as follows.

[0071] Table 1. Clinical information of subjects who completed single-cell sequencing

[0072]

[0073] After quality control and elimination of cells with too few genes or too many mitochondrial genes, a total of 29,620 cells were included in the analysis, including 4,233 cells in the CA group, 6,896 cells in the CVA group, 6,795 cells in the EB group, and 11,716 cells in the HC group (Table 1). The average number of genes in these cells was 2,774, and the average count was 13,511. After batch correction, dimensionality reduction clustering, SingleR automated cell type annotation of cell clusters, and further identification of cell types through cell-specific marker genes, a total of 6 major cell types were identified ( Figure 1 A, 1B), including B cells (expressing CD19, CD79A), T cells (expressing CD2, CD3D), epithelial cells (expressing KRT8, KRT19), mast cells (expressing TPSB2, CPA3), dendritic cells (expressing CD1C) and monocytes / macrophages (expressing CD163) ( Figure 1 D). Based on the expression of genes such as FABP4, SPP1, and FCN1, monocytes / macrophages can be further divided into FABP4+ macrophages (high expression of FABP4 and MARCO), SPP1+ macrophages (high expression of SPP1, low or no expression of FABP4 and FCN1), and FCN1+ monocytes (high expression of FCN1 and VCAN, low or no expression of FABP4 and SPP1). From the distribution of cell types, each sequenced sample can identify the above cell types ( Figure 1 C).

[0074] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing an induced sputum single cell suspension, characterized in that: The steps include: 1) obtaining a filtered sputum suspension to be tested; 2) Low temperature centrifugation; 3) After centrifugation of the sample, the supernatant was removed without destroying the cell pellet; 5) Add DPBS without calcium and magnesium ions to the cell pellet, mix gently with a pipette, and repeatedly invert the tube to resuspend the cell pellet; 6) Filter and collect the filtrate in a new centrifuge tube; 7) performing low-temperature centrifugation on the filtrate collected in the previous step, and removing the supernatant without destroying the cell pellet; 8) Add an appropriate amount of DPBS without calcium and magnesium ions to the cell pellet in the previous step, mix gently with a pipette, and gently pipette 10-15 times or until the cells are completely suspended; 9) Adjust the suspension volume to obtain the target cell concentration, that is, a single-cell suspension.

2. The preparation method according to claim 1, characterized in that: The parameters of the low-temperature centrifugation are: 1200±10 rpm, 4°C, and centrifugation for 4-6 min.

3. The preparation method according to claim 1, characterized in that: The target cell concentration is 700-1200 cells / ul.

4. The preparation method according to claim 1, characterized in that: In step 6), the pore size of the filter used for filtration is 30±5 um.

5. The preparation method according to any one of claims 1 to 4, characterized in that: In step 6), when using a filter, the filter should be first wetted with DPBS without calcium and magnesium ions. After the cell suspension is filtered, 500-1000ul of DPBS without calcium and magnesium ions is added to the filter to collect as many filtered cells as possible.

6. The induced sputum single cell suspension prepared according to the method according to any one of claims 1 to 5.

7. Use of the method for preparing induced sputum single cell suspension according to any one of claims 1 to 5 in a method for induced sputum single cell transcriptome sequencing.

8. A method for single-cell transcriptome sequencing of induced sputum, characterized in that: The following steps are involved: The induced sputum single cell suspension prepared according to the method according to any one of claims 1 to 5 is used for scRNA-seq library construction and sequencing.

9. The method for single-cell transcriptome sequencing of induced sputum according to claim 8, characterized in that: The scRNA-seq library construction and sequencing include the following steps: A) GEM generation and labeling; Single cell suspension of induced sputum was isolated and labeled by reverse transcription; B) Post GEM-RT purification and cDNA amplification; C) Library construction and quantification; D) Sequencing the constructed library.

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