A method for single-cell transcriptome sequencing

Abstract

The present invention provides a method for single-cell transcriptome sequencing. Specifically, the method of the present invention adopts the "one-tube method" to complete the sample lysis, reverse transcription, PCR amplification and product purification steps in the same reaction vessel, thereby improving the sensitivity of amplification of low-abundance RNA in the cell transcriptome and RNA in special structures, reducing the loss of transcriptome information, and improving the efficiency of single-cell transcriptome amplification and sequencing. In addition, the method of the present invention is applicable to transcriptome sequencing of fixed single-cell samples and live fresh single-cell samples. The present invention also provides a kit for single-cell transcriptome sequencing and a single-cell transcriptome sequencing device, which realizes more convenient, rapid, sensitive and complete sequencing of single-cell transcriptomes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for single-cell transcriptome sequencing. Background Art

[0002] Single-cell transcriptome sequencing is a new technology for amplifying and sequencing transcriptomes at the single-cell level. Its principle is to amplify the trace transcriptome RNA of isolated single cells and then perform high-throughput sequencing. The single-cell transcriptome sequencing methods currently widely used in the market are: HT Kit and 10XGenomics single-cell transcriptome sequencing platform. The HT Kit can obtain full-length cDNA from a single cell and perform downstream amplification and library construction and sequencing, but the throughput is very low and the price is expensive. The 10XGenomics single-cell transcriptome sequencing platform can perform single-cell transcriptome sequencing on tens of thousands of cells at a time, but the principle of this method means that only the 3' end information of the transcriptome can be obtained. Moreover, both of the above methods can only perform single-cell transcriptome sequencing on fresh, living single cells.

[0003] In the research process, we often encounter situations where the proportion of target cells in the entire single-cell suspension is very low, and it takes a long time to screen. In this process, the activity of single cells will be weakened and the transcripts will change. If the cells can be fixed before screening so that the transcripts no longer change, it will give the experimenter more time to screen cells and obtain more accurate single-cell transcript information.

[0004] The research results published so far, such as Elliot R Thomsen et al., Nat Methods. 2016; 13(1): 87-93 and Johannes W Bagnoli et al., Nat Commun. 2018; 9(1): 2937, indicate that fixed single-cell samples can also be used to construct single-cell transcriptome libraries and perform subsequent library construction sequencing. However, in the process of transcriptome amplification for fixed single-cell samples mentioned in Elliot R Thomsen et al., Nat Methods. 2016; 13(1): 87-93, the released mRNA needs to be purified, and in the process of transcriptome amplification for fixed single-cell samples mentioned in Johannes W Bagnoli et al., Nat Commun. 2018; 9(1): 2937, the reverse transcription product needs to be purified. Purification of trace amounts of mRNA and cDNA will result in the loss of low-abundance transcript information.

[0005] Therefore, there is an urgent need to develop a new single-cell transcriptome sequencing method in this field. Summary of the invention

[0006] The purpose of the present invention is to provide a method for single-cell transcriptome amplification, library construction and sequencing.

[0007] In a first aspect of the present invention, a single cell transcriptome sequencing method is provided, the method mainly comprising the following steps:

[0008] (a) providing a single cell sample and placing it in a reaction container;

[0009] (b) lysing the sample (i.e., decrosslinking) using a buffer containing proteinase K to lyse the product;

[0010] (c) performing a reverse transcription reaction using the cleavage product as a template to obtain a reverse transcription product containing cDNA;

[0011] (d) performing PCR amplification on the reverse transcription product to obtain an amplified product;

[0012] (e) purifying the amplified product to obtain a purified amplified product; and

[0013] (f) constructing a library and sequencing the purified amplification product to obtain single-cell transcriptome sequencing data;

[0014] Wherein, steps (b), (c) and (d) and the steps therebetween do not include nucleic acid purification operations;

[0015] Furthermore, steps (b), (c) and (d) are all carried out in the same reaction vessel.

[0016] In another preferred embodiment, the step (e) is also carried out in the same reaction vessel.

[0017] In another preferred embodiment, the single cell is a fresh single cell or a fixed single cell.

[0018] In another preferred embodiment, the single cell is selected from human kidney embryo cells, neuronal cells or other types of cells.

[0019] In another preferred embodiment, in step (b), the amount of proteinase K used is 0.2-1.5 mg / ml (μg / μl), that is, (0.4-4.5 μg) proteinase K / (2-3 μl) cleavage reaction volume.

[0020] In another preferred embodiment, in step (b), the buffer is TE buffer.

[0021] In another preferred embodiment, the TE buffer comprises the following components: 10 mM Tris·HCl, 1 mM EDTA and a solvent (eg, water).

[0022] In another preferred embodiment, the pH value of the TE buffer is 7.0-8.8, preferably about 8.0.

[0023] In another preferred embodiment, the reaction temperature of cleavage (decrosslinking) in step (b) is 50-70°C, preferably 55-65°C, and most preferably about 56°C.

[0024] In another preferred embodiment, the reaction time of cleavage (decrosslinking) in step (b) is 10 minutes to 3 hours, preferably 0.25 to 2 hours, more preferably 0.5 to 1.5 hours, and most preferably about 1 hour.

[0025] In another preferred embodiment, the cleavage product contains released RNA.

[0026] In another preferred embodiment, between steps (b) and (c), the method further includes: performing an inactivation treatment on the cleavage product, thereby inactivating proteinase K.

[0027] In another preferred embodiment, the inactivation treatment includes heating at 95±3°C for a period of time (eg, 5-30 minutes).

[0028] In another preferred embodiment, the reaction system of the reverse transcription reaction in step (c) contains macromolecular polyethylene glycol, and the macromolecular polyethylene glycol is selected from the following group: PEG6000, PEG7000, PEG8000, PEG9000 or a combination thereof.

[0029] In another preferred embodiment, the reaction system of the reverse transcription reaction in step (c) contains PEG8000.

[0030] In another preferred embodiment, the final concentration of PEG8000 in the reaction system is 6-10% (v / v), preferably 7.5%.

[0031] In another preferred embodiment, the reaction system further includes TSO primers, oligo-dT primers, and other necessary reagents required for the reverse transcription reaction.

[0032] In another preferred embodiment, the 3' end of the TSO primer contains rGrG+G modification.

[0033] In another preferred embodiment, the 3' end of the oligo-dT primer contains a poly T modification.

[0034] In another preferred embodiment, other necessary reagents required for the reverse transcription reaction include reverse transcriptase, reaction buffer, reducing agent (such as DTT), and RNase inhibitor.

[0035] In another preferred embodiment, the cleavage or step (b) is carried out in a first reaction system with a volume of V1.

[0036] In another preferred embodiment, the reverse transcription or step (c) is carried out in a second reaction system with a volume of V2.

[0037] In another preferred embodiment, the PCR reaction or step (d) is carried out in a third reaction system with a volume of V3.

[0038] In another preferred example, V1<V2<V3.

[0039] In another preferred embodiment, V2 / V1=1.5-4, preferably 2-3.

[0040] In another preferred embodiment, V3 / V2=1.5-4, preferably 2-3.

[0041] In another preferred embodiment, the V1 is 2-5 μl.

[0042] In another preferred embodiment, the V2 is 5-15 μl.

[0043] In another preferred embodiment, the V3 is 15-30 μl.

[0044] In another preferred embodiment, in step (e), magnetic beads are used for purification.

[0045] In a second aspect of the present invention, a kit for single-cell transcriptome sequencing is provided, the kit comprising a cell lysis reagent, a reverse transcription reagent, a PCR amplification reagent and a reaction container.

[0046] In another preferred embodiment, the kit further comprises a label and instructions.

[0047] In another preferred embodiment, the kit optionally comprises a reagent for purifying the DNA amplification product.

[0048] In another preferred embodiment, the cell lysis reagent includes proteinase K and TE buffer.

[0049] In another preferred embodiment, the cell lysis reagent is TE buffer containing proteinase K.

[0050] In another preferred embodiment, the proteinase K and TE buffer are provided independently.

[0051] In another preferred embodiment, the proteinase K and TE buffer may be solid preparations or liquid preparations.

[0052] In another preferred embodiment, the working concentration of the proteinase K is 0.2-1.5 mg / ml (μg / μl), that is, (0.4-4.5 μg) proteinase K / (2-3 μl) cleavage reaction volume.

[0053] In another preferred embodiment, the TE buffer comprises the following components: 10 mM Tris·HCl, 1 mM EDTA and a solvent (eg, water).

[0054] In another preferred embodiment, the pH value of the TE buffer is 7.0-8.8, preferably about 8.0.

[0055] In another preferred example, the reverse transcription reagent includes PEG8000, TSO primer, oligo-dT primer, reverse transcriptase, reaction buffer, reducing agent (such as DTT), and RNase inhibitor.

[0056] In another preferred embodiment, the working concentration of PEG8000 is a final concentration of 6-10% (v / v), preferably 7.5%.

[0057] A third aspect of the present invention provides a single-cell transcriptome sequencing device, the device comprising:

[0058] (M1) a sample loading module, wherein the sample loading module is configured to: place a single cell sample in a reaction container, wherein the reaction container is a first container;

[0059] (M2) a cleavage module, wherein the cleavage module is configured to: add a buffer containing proteinase K into the reaction container to cleave the sample to obtain a cleavage product;

[0060] (M3) a reverse transcription module, wherein the reverse transcription module is configured to: add a reverse transcription reaction reagent into the reaction container to perform a reverse transcription reaction on the cleavage product to obtain a reverse transcription product containing cDNA;

[0061] (M4) a PCR module, wherein the PCR module is configured to: add a PCR reaction reagent into the reaction container to perform PCR amplification on the reverse transcription product to obtain an amplified product;

[0062] (M5) a purification module, wherein the purification module is configured to: purify the amplification product in the reaction container and transfer the purified amplification product to a second container, wherein the second container is different from the first container;

[0063] (M6) a sequencing module, the sequencing module being configured to: sequence the purified amplification product in the second container;

[0064] Wherein, the reaction container is the same reaction container.

[0065] In another preferred embodiment, the sequencing device further includes: (M7) a control module for controlling the operation of the modules (M1) a sample loading module, (M2) a cleavage module, (M3) a reverse transcription module, (M4) a PCR module, (M5) a purification module and (M6) a sequencing module.

[0066] In another preferred example, the control module controls the following modules in the single-cell transcriptome sequencing device to operate sequentially: (M1) loading module, (M2) lysis module, (M3) reverse transcription module, (M4) PCR module, (M5) purification module and (M6) sequencing module, that is, they operate in the order of (M1) → (M2) → (M3) → (M4) → (M5) → (M6).

[0067] In another preferred example, the (M2) lysis module further includes an inactivation submodule, and the inactivation submodule is configured to inactivate the lysis product to obtain an inactivated lysis product.

[0068] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The results of DNA product detection of negative control amplified by single-cell transcriptome amplification technology LF-seq-v1 are shown. The peak graph on the left shows the size distribution of DNA products, and the gel capillary electrophoresis results of DNA products are shown on the right.

[0070] Figure 2 The results of DNA product detection of the positive control amplified by the single-cell transcriptome amplification technology LF-seq-v1 are shown. The peak graph on the left shows the size distribution of the DNA product, and the gel capillary electrophoresis results of the DNA product are shown on the right.

[0071] Figure 3 The results of DNA product detection of fresh 293T cells amplified by single-cell transcriptome amplification technology LF-seq-v1 are shown. The peak graph on the left shows the size distribution of DNA products, and the gel capillary electrophoresis results of DNA products are shown on the right.

[0072] Figure 4The results of DNA product detection of fixed 293T cells amplified by the single-cell transcriptome amplification technology LF-seq-v1 are shown. The peak graph on the left shows the size distribution of the DNA products, and the gel capillary electrophoresis results of the DNA products on the right are shown.

[0073] Figure 5 Shows the use Statistical results of gene sequencing after single-cell transcriptome amplification using HT Kit and LF-seq-v1. Group T indicates that the sample is a living single 293T cell and is HTKit for single-cell transcriptome amplification. The Fresh group indicates that the sample is a living single 293T cell, and the single-cell transcriptome amplification is performed using this technology LF-seq-v1. The Fixed group indicates that the sample is a fixed single 293T cell, and the single-cell transcriptome amplification is performed using this technology LF-seq-v1. The numbers indicate the number of genes.

[0074] Figure 6 The GO analysis results of 6580 genes are shown.

[0075] Figure 7 Shows the use PCR analysis results of histone genes H1-4 in amplified samples after amplification of live single 293T cells using HT Kit and fixed single 293T cells using LF-seq-v1. The corresponding relationship between the markers and samples in the figure is: Ladder: 250bp DNA ladder (TransGen); HT-1: T_sample_1; HT-2: T_sample_2; HT-3: T_sample_3; FL1: fixed_sample_1; FL2: fixed_sample_2; FL3: fixed_sample_3. HT-1 (T_sample_1), HT-2 (T_sample_2), and HT-3 (T_sample_3) are samples amplified using Samples of HT Kit; FL1 (fixed_sample_1), FL2 (fixed_sample_2), and FL3 (fixed_sample_3) are samples using LF-seq-v1 of the present invention.

[0076] Figure 8 Shows the use PCR analysis results of low-abundance membrane protein and extracellular matrix protein genes PECAM1, PDGFRB, CHAT, and GFAP in amplified samples after amplification of live single 293T cells using HT Kit and fixed single 293T cells using LF-seq-v1. The corresponding relationship between the markers and samples in the figure is: Ladder: 250bp DNA ladder (TransGen); HT-2: T_sample_2; FL1: fixed_sample_1; FL2: fixed_sample_2; FL3: fixed_sample_3. HT-2 (T_sample_2) is amplified using Samples of HT Kit; FL1 (fixed_sample_1), FL2 (fixed_sample_2), and FL3 (fixed_sample_3) are samples using LF-seq-v1.

[0077] Fig. 9 The immunohistochemical staining results of PECAM1, PDGFRB, CHAT and GFAP gene expression corresponding to the 293T samples used are shown. DETAILED DESCRIPTION

[0078] After extensive and in-depth research, the inventors have discovered and developed for the first time a single-cell transcriptome amplification sequencing method applicable to both living cells and fixed cells. By optimizing reagents and streamlining experimental steps, sample lysis, reverse transcription, PCR amplification, and product purification steps can be completed in a single reaction vessel, thereby significantly improving the sensitivity of amplification of low-abundance RNA in the cell transcriptome and RNA in special structures, reducing the loss of transcriptome information, and improving the efficiency of single-cell transcriptome amplification and sequencing. On this basis, the present invention was completed.

[0079] the term

[0080] Single-cell transcriptome sequencing

[0081] Single-cell transcriptome sequencing is a new technology that amplifies and sequences transcriptomes at the single-cell level. Its principle is to amplify the trace transcriptome RNA of isolated single cells and then perform high-throughput sequencing.

[0082] In the early stage, single-cell transcriptomics mainly relied on three methods: SMART-seq2 technology, 10×genomics technology and Andeplete technology.

[0083] SMART-seq2 technology can be used for single-cell mRNA sequencing. It has high requirements for RNA quality. RNA degradation will cause loss of information at the 5' end. It can obtain full-length RNA amplification cDNA products, which are all used for library construction and sequencing after interruption. Anydeplete technology can be used for single-cell mRNA and LncRNA sequencing. It does not have high requirements for RNA quality and can be used for sequencing degradable samples. 10×genomics technology can be used for single-cell mRNA sequencing. It has high requirements for RNA quality. RNA degradation will cause loss of information at the 5' end. It can separate 500 to 10,000 cells at a time and can sequence the trace mRNA in the separated single cells after efficient amplification. It can only measure a small sequence at the 3' end.

[0084] Proteinase K

[0085] Proteinase K is a powerful proteinase isolated from Candida albicans. It has a high specific activity and is a key reagent for DNA extraction. The enzyme is active in a wide pH range (4-12.5) and high temperature (50-70°C) and is used to separate plasmid or genomic DNA and RNA. In DNA extraction, its main function is to enzymatically hydrolyze histones bound to nucleic acids, freeing DNA in the solution, and then extracting it using different methods to remove impurities and collect DNA.

[0086] In the method of the present invention, TritonX-100 conventionally used in the cell lysis step is replaced with proteinase K, which can achieve the purpose of cell lysis and at the same time, perform a cross-linking reaction, digest proteins, and release transcriptome RNA.

[0087] PEG

[0088] In the present invention, the inventors unexpectedly discovered that the use of a specific type of PEG can efficiently reverse transcribe the cleavage product, and when present in a preferred content, it can remain in the reaction vessel without purification, without adversely affecting subsequent PCR amplification and product purification.

[0089] In the present invention, the preferred PEG is PEG6000-10000, preferably PEG7000-PEG9000, that is, PEG with a molecular weight of 7000 to 9000. For example, PEG8000 (i.e. polyoxyethanol 8000, also known as polyoxyethylene 8000).

[0090] In a preferred embodiment of the present invention, PEG8000 is used in the reverse transcription step (instead of betaine in the conventional method), and the optimal reaction concentration of PEG8000 is determined by repeated experiments. The concentration of PEG8000 used in the method of the present invention ensures that the reverse transcription reaction is carried out efficiently, and it is retained in the reaction solution without removal and will not affect the subsequent PCR reaction. Therefore, in the method of the present invention, the purification step after reverse transcription is omitted, the overall operation process is simplified, and the loss of low-abundance genes caused by the purification step is reduced.

[0091] Kits for single-cell transcriptome sequencing

[0092] As described herein, the "kit for single-cell transcriptome sequencing" refers to a kit for sample preparation before single-cell transcriptome sequencing, comprising the cell lysate, reverse transcription reagent, PCR amplification reagent and a reaction container described in the present invention, and the kit should also include instructions or labels marked with the method of the present invention. In addition, the kit may optionally include a DNA amplification product purification reagent.

[0093] In a preferred embodiment of the present invention, the cell lysate includes proteinase K and TE buffer, which can be a TE buffer containing proteinase K (the working concentration of proteinase K is 0.2-1.5 mg / ml (μg / μl), i.e. (0.4-4.5 μg) proteinase K / (2-3 μl) lysis reaction volume), or a proteinase K preparation and a TE buffer preparation provided separately. The proteinase K preparation and TE buffer preparation can be solid preparations or liquid preparations. When used, the proteinase K preparation is added to the TE buffer to prepare a cell lysate with a working concentration of 0.2-1.5 mg / ml (or μg / μl).

[0094] In a preferred embodiment of the present invention, the TE buffer is prepared according to the following formula: 10 mM Tris·HCl, 1 mM EDTA and a solvent (such as water). In another preferred embodiment, the pH value of the TE buffer is 7.0-8.8, preferably about 8.0.

[0095] In a preferred embodiment of the present invention, the reverse transcription reagent comprises PEG8000, TSO primer, oligo-dT primer, reverse transcriptase, reaction buffer, reducing agent (such as DTT), and RNase inhibitor. The working concentration of the PEG8000 is 6-10% (v / v), preferably 7.5%.

[0096] In a preferred example of the present invention, the PCR amplification reagent is a reagent used in conventional PCR, including a front primer, a rear primer, a PCR amplification enzyme, dNTP and a reaction buffer.

[0097] Single-cell transcriptome sequencing device

[0098] The present invention also provides a single-cell transcriptome sequencing device that can be manually controlled or automated by an instrument. After adding a single-cell sample, the device can perform cell lysis (cross-linking removal), reverse transcription, PCR amplification, DNA amplification product purification, and sequencing analysis to obtain single-cell transcriptome data. The device can realize large-scale single-cell transcriptome data collection and is suitable for single-cell transcriptome information library construction.

[0099] The device of the present invention comprises (M1) a sample loading module, (M2) a lysis module, (M3) a reverse transcription module, (M4) a PCR module, (M5) a purification module and (M6) a sequencing module, and can realize the functions of cell lysis, reverse transcription, PCR amplification, DNA product purification and sequencing.

[0100] In another preferred embodiment, the sequencing device further includes: (M7) a control module for controlling the operation of the modules (M1) a sample loading module, (M2) a cleavage module, (M3) a reverse transcription module, (M4) a PCR module, (M5) a purification module and (M6) a sequencing module.

[0101] In another preferred example, the control module controls the following modules in the single-cell transcriptome sequencing device to operate sequentially: (M1) loading module, (M2) lysis module, (M3) reverse transcription module, (M4) PCR module, (M5) purification module and (M6) sequencing module, that is, they operate in the order of (M1) → (M2) → (M3) → (M4) → (M5) → (M6).

[0102] In another preferred example, the (M2) lysis module further includes an inactivation submodule, and the inactivation submodule is configured to inactivate the lysis product to obtain an inactivated lysis product.

[0103] The device of the present invention may further include a computer, a data storage device, a cooling, heating and constant temperature incubation device and other connecting components to form a complete device.

[0104] The main advantages of the present invention are:

[0105] (1) The single-cell transcriptome sequencing method provided by the present invention is applicable to both fresh living single-cell samples and fixed single-cell samples.

[0106] (2) In the method described in the present invention, the lysis, reverse transcription, and PCR amplification steps are all completed in the same reaction vessel, which reduces the loss of sample information caused by sample transfer during the implementation process and greatly preserves the integrity of the single-cell transcriptome.

[0107] (3) In the method described in the present invention, the TE buffer used in the lysis step is suitable for proteinase K lysis and is particularly compatible with subsequent PCR.

[0108] (4) In the method described in the present invention, the purification step of removing macromolecules (such as PEG8000) in the reaction system after sample reverse transcription is omitted, thereby reducing the loss of low-abundance genes.

[0109] (5) The method provided by the present invention improves the amplification sensitivity for low-abundance RNA and RNA in some special structures (expressing histones, membrane proteins and some extracellular matrix proteins).

[0110] In summary, the present invention provides an optimized single-cell transcriptome amplification sequencing method with simple steps, high sensitivity, good integrity and wider applicability.

[0111] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0112] Unless otherwise specified, the materials, reagents (including reverse transcription primers, TSO primers), instruments, etc. used in the examples can be obtained from commercial sources.

[0113] Example 1 Experimental process of single-cell transcriptome amplification technology LF-seq-v1

[0114] Experimental procedures

[0115] 1. Release of single-cell RNA

[0116] 1.1 Prepare cell lysis buffer (for 8 reactions): 15 μl TE + 1 μl 20 mg / ml PK (in TE buffer). Dispense into 0.2 ml EP tubes at 2 μl / tube.

[0117] 1.2 Single cell sample preparation: Use a single cell acquisition device to place fixed (fixed with 4% paraformaldehyde) or live single cells into the above lysis solution. Prepare a positive control group (10pg 293T total RNA) and a negative control group (a small amount of supernatant aspirated from the cell suspension, without cells, composed of PBS + 0.04% BSA).

[0118] 1.3 Lysis (cross-linking removal): Incubate the cells and lysis buffer mixture at 56°C for 1 hour, then add 1 μl oligo(dT) 30 Primer + 1 μl dNTP (final system is 4.3 μl = 2 μl lysis buffer + 1 μl oligo (dT) 30 Primer+1 μl dNTP+0.3 μl 293T total RNA / single cell (in PBS+0.04% BSA) / PBS+0.04% BSA), react at 56°C for 2 minutes in a thermal cycler, and inactivate at 95°C for 10 minutes.

[0119] 2. Reverse Transcription

[0120] 2.1 Prepare the reverse transcription reaction solution according to the formula listed in Table 1.

[0121] Table 1

[0122]

[0123] The TSO primer sequence is as follows:

[0124] 5'-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3' (SEQ ID NO.: 1).

[0125] 2.2 Add 5.7 μl of the reverse transcription reaction solution in step 2.1 to the cleavage (decrosslinking) product in step 1.3 to obtain a final reaction volume of 10 μl. After mixing, perform the reverse transcription reaction according to the following steps (as shown in Table 2):

[0126] Table 2

[0127]

[0128] After the reaction is completed, a product containing single-stranded cDNA is obtained. Compared with RNA, this cDNA has an additional sequence on TSO as the site for the next round of PCR primer binding.

[0129] 3. PCR Amplification

[0130] 3.1 Prepare the PCR reaction solution according to the formula shown in Table 3 below:

[0131] Table 3

[0132]

[0133] The IS PCR primers sequences are as follows:

[0134] 5'-AAGCAGTGGTATCAACGCAGAGT-3' (SEQ ID NO.:2).

[0135] 3.2 Add 15 μl of the PCR reaction solution in step 3.1 to the reaction system in step 2.2 to obtain a final volume of 25 μl of the reaction solution. Use a PCR instrument to amplify the transcriptome according to the process in Table 4 below. The number of amplification cycles is adjusted according to the type of sample.

[0136] Table 4

[0137]

[0138] 4. DNA Purification

[0139] Use Ampure XP beads to purify the PCR product in step 3.2. The specific steps are as follows:

[0140] (1) Before starting the purification step, equilibrate Ampure XP beads at room temperature for 15 minutes and then vortex for a few seconds.

[0141] (2) Add 20 μl of Ampure XP beads (ratio 1:0.8) to each PCR sample (25 μl) obtained in step 3.2 and pipette up and down 10 times or until the solution is homogenous.

[0142] (3) Incubate at room temperature for 8 minutes to allow DNA to bind to the beads.

[0143] (4) Place the PCR tube containing the PCR product and magnetic beads in step (3) on a magnet rack and let it stand for 5 minutes or until the solution is clear, allowing the beads to aggregate on one side of the tube.

[0144] (5) While keeping the PCR tube in step (4) on the magnet rack, carefully remove the supernatant without disturbing the magnetic beads.

[0145] (6) Add 200 μl of 80% (vol / vol) ethanol solution to the PCR tube in step (5), incubate with the sample at room temperature for 30 seconds to wash the magnetic beads, and then remove the ethanol. Repeat this step.

[0146] (7) Remove any residual ethanol, allow the beads to dry completely, and place the PCR tube in step (6) at room temperature for 5 minutes, or until a small crack appears on the surface of the beads.

[0147] (8) Remove the PCR tube in step (7) from the magnetic rack, add 17 μl of EB solution (or nuclease-free water), and mix ten times to resuspend the beads.

[0148] (9) Incubate the PCR tube in step (8) at room temperature for 2 minutes.

[0149] 10) Place the PCR tube from step (9) on a magnet stand and let stand for 2 minutes until the solution becomes clear and the beads aggregate on one side of the tube.

[0150] (11) Set the pipette volume to 15 μl, collect the supernatant, avoid sucking up the beads, and transfer the supernatant to another 0.2 ml thin-walled PCR tube.

[0151] Care should be taken not to increase the volume of beads above a 1:0.8 ratio during the purification step. Using less than the standard amount of beads ensures that substrate dimer carryover is kept to a minimum.

[0152] Experimental Results

[0153] In the Fragment Analyzer TM fully automated capillary electrophoresis system, the FragmentAnalyzer Large Fragment Analysis Kits (Cat. No.: DNF-464-500) were used to detect the size distribution of the extracted DNA products. The main peak of a good library should be around 1.5-2 kb.

[0154] The negative control test results are as follows Figure 1 shown.

[0155] The positive control test results are as follows Figure 2 shown.

[0156] The test results of fresh 293T cells are as follows Figure 3 shown.

[0157] The detection results of fixed 293T cells are as follows Figure 4 shown.

[0158] The results showed that this method can well amplify the transcriptome library from the positive control (10pg 293T total RNA), fresh 293T single cell samples, and fixed 293T single cell samples, but will not non-specifically amplify the transcriptome from the negative control (293T cell suspension (without cells)).

[0159] The above-mentioned single-cell transcriptome amplification products can be further sequenced.

[0160] Example 2 Single-cell transcriptome amplification technology LF-seq-v1 and HT Kit Comparison

[0161] The single-cell transcriptome amplification technology LF-seq-v1 described in the present invention and the commercially available HT Kit for single-cell transcriptome amplification. The specific operation steps of LF-seq-v1 amplification technology are as described in Example 1; The single-cell transcriptome amplification of the HT Kit was performed according to the protocol in the kit instructions. The samples used in the HT Kit method are live single 293T cells; the samples used in the LF-seq-v1 amplification technology are live single 293T cells and fixed single 293T cells. After amplification, the amplified samples were sequenced, and the sequencing results are as follows: Figure 5 shown.

[0162] The results showed that the genes obtained from fixed single 293T cells and living single 293T cells using the single-cell transcriptome amplification technology LF-seq-v1 described in the present invention were significantly higher than those obtained using traditional The HT Kit retrieves 6,580 more genes from living single 293T cells.

[0163] GO analysis was performed on these 6580 genes (e.g. Figure 6 As shown in Figure 2, most of these genes are membrane proteins and extracellular matrix proteins, as well as highly expressed histones. Since these proteins need to be modified by the endoplasmic reticulum and Golgi apparatus after translation, the localization of the mRNA corresponding to these genes may have some structural peculiarities, making it difficult to release them using the traditional triton X-100 cleavage method. However, the cross-linking method using proteinase K can release them well.

[0164] Furthermore, PCR and immunohistochemistry methods were used to further verify that the extra genes were indeed expressed. Figure 7 As shown, use The HT Kit could not obtain transcripts of the abundant histone genes H1-4, but the method described in this article could obtain them well. In addition, by PCR ( Figure 8 ) and immunohistochemistry ( Fig. 9 ) method proves the use of The HT Kit cannot obtain transcripts of low-abundance membrane proteins and extracellular matrix proteins (such as PECAM1, PDGFRB, CHAT, and GFAP), but this method can obtain them well. Figure 8 The results of gel electrophoresis after PCR show the results of gel electrophoresis of DNA amplification products obtained using two different methods. It can be seen from the figure that the amount of PECAM1, PDGFRB, CHAT and GFAP transcripts in the products obtained using the method described in this article is significantly higher than that using Products obtained with HT Kit. Fig. 9The expression of PECAM1, PDGFRB, CHAT and GFAP genes in the used 293T cells is shown. It can be seen from the figure that the expression of PECAM1, PDGFRB, CHAT and GFAP in 293T cells is at a low level.

[0165] The above experiments prove that the single-cell amplification method LF-seq-v1 described in this article can be used for both live and fixed single-cell amplification, and compared with existing methods, it reduces the loss of low-abundance transcripts and improves the sensitivity of obtaining low-abundance transcripts.

[0166] discuss

[0167] In the single-cell transcriptome amplification method described in the present invention, the cell lysis system is changed from triton X-100 commonly used in the prior art to proteinase K lysis, and the lysis buffer is replaced with TE buffer, so that the technology described in the present invention can be used for the amplification of living single cells as well as fixed single cells. In addition, the lysis method using proteinase K can also release RNA in some special structures in the cell, so that more genes can be detected.

[0168] In the current prior art, after the RNA release is completed, the mRNA is usually purified, or the PCR product is purified after the first round of PCR is completed. The purification step increases the complexity and difficulty of the experiment and causes the loss of some low-abundance RNA. The entire reaction of the single-cell transcriptome amplification method of the present invention is completed in the same system, and the experiment is more convenient. Subsequent data analysis further shows that the single-cell transcriptome amplification method of the present invention is more sensitive in obtaining low-abundance transcripts.

[0169] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences <120> A method for single-cell transcriptome sequencing <130> P2020-1573 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <400> 1 aagcagtggt atcaacgcag agtacatggg 30 <210> 2 <211> twenty three <212> DNA <213> Artificial Sequence <400> 2 aagcagtggt atcaacgcag agt 23

Claims

1. A single-cell transcriptome sequencing method, characterized in that: The method mainly comprises the following steps: (a) providing a single cell sample and placing it in a reaction container, wherein the single cell is a fresh single cell or a fixed single cell, wherein the fixed single cell is fixed with paraformaldehyde; (b) using a buffer containing proteinase K to lyse the sample, i.e., to decrosslink, thereby lysing the product, wherein the amount of proteinase K used is 0.2-1.5 μg / μl, i.e., 0.4-4.5 μg proteinase K / 2-3 μl lysis reaction volume; the buffer is TE buffer; (c) using the cleavage product as a template to perform a reverse transcription reaction to obtain a reverse transcription product containing cDNA, wherein the reaction system of the reverse transcription reaction contains macromolecular polyethylene glycol, the macromolecular polyethylene glycol is selected from PEG8000, and the final concentration of the macromolecular polyethylene glycol in the reaction system is 8% (v / v); (d) performing PCR amplification on the reverse transcription product to obtain an amplified product; (e) purifying the amplified product to obtain a purified amplified product; and (f) constructing a library and sequencing the purified amplification product to obtain single-cell transcriptome sequencing data; Wherein, steps (b), (c) and (d) and the steps therebetween do not include nucleic acid purification operations; Furthermore, steps (b), (c), (d) and (e) are all carried out in the same reaction vessel; and between steps (b) and (c), the method further comprises: performing an inactivation treatment on the cleavage product, thereby inactivating proteinase K.

2. The method according to claim 1, characterized in that The fixed single cells were fixed with 4% paraformaldehyde.

3. The method according to claim 1, characterized in that The reaction temperature of the cracking in step (b) is 50-70°C.

4. The method according to claim 3, characterized in that The reaction temperature of the cracking in step (b) is 55-65°C.

5. The method according to claim 1, characterized in that The reaction time of the cleavage in step (b) is 10 minutes to 3 hours.

6. The method according to claim 5, characterized in that The reaction time of the cleavage in step (b) is 0.5-1.5 hours.

7. The method according to claim 1, characterized in that The lysis or step (b) is performed in a first reaction system with a volume of V1, the reverse transcription or step (c) is performed in a second reaction system with a volume of V2, and the PCR reaction or step (d) is performed in a third reaction system with a volume of V3, and V1<V2<V3.

8. A single-cell transcriptome sequencing device, characterized in that: The device comprises: (M1) a sample loading module, wherein the sample loading module is configured to: place a single cell sample in a reaction container, wherein the reaction container is a first container, wherein the single cell is a fresh single cell or a fixed single cell, wherein the fixed single cell is fixed with paraformaldehyde; (M2) a cleavage module, wherein the cleavage module is configured to: add a buffer containing proteinase K into the reaction container to cleave the sample to obtain a cleavage product; wherein the amount of proteinase K is 0.2-1.5 μg / μl, i.e., 0.4-4.5 μg proteinase K / 2-3 μl cleavage reaction volume; the buffer is TE buffer; and the (M2) cleavage module further includes an inactivation submodule, wherein the inactivation submodule is configured to inactivate the cleavage product to obtain an inactivated cleavage product; (M3) a reverse transcription module, wherein the reverse transcription module is configured to: add a reverse transcription reaction reagent into the reaction container to perform a reverse transcription reaction on the cleavage product to obtain a reverse transcription product containing cDNA; wherein the reverse transcription reagent comprises macromolecular polyethylene glycol, the macromolecular polyethylene glycol is selected from PEG8000, and the final concentration of the macromolecular polyethylene glycol in the reaction system is 8% (v / v); (M4) a PCR module, wherein the PCR module is configured to: add a PCR reaction reagent into the reaction container to perform PCR amplification on the reverse transcription product to obtain an amplified product; (M5) a purification module, wherein the purification module is configured to: purify the amplification product in the reaction container and transfer the purified amplification product to a second container, wherein the second container is different from the first container; (M6) a sequencing module, the sequencing module being configured to: sequence the purified amplification product in the second container; Wherein, the reaction container is the same reaction container; In addition, the sequencing device further includes: (M7) a control module, which is used to control the operation of the modules (M1) loading module, (M2) lysis module, (M3) reverse transcription module, (M4) PCR module, (M5) purification module and (M6) sequencing module, and operates in the order of (M1) → (M2) → (M3) → (M4) → (M5) → (M6).

9. The sequencing device according to claim 8, wherein: In the (M1) loading module, the fixed single cells are fixed with 4% paraformaldehyde.

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