CN111394801A - Construction method of multiple single-cell simplified representative methylation library based on Illumina sequencing platform - Google Patents
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Abstract
The invention provides a method for constructing a multiple single cell simplified representative methylation library based on an Illumina sequencing platform, which comprises the following steps: (1) single cell lysis or trace DNA extraction and purification; (2) digesting with restriction enzyme Msp I; (3) end repairing and adding A at the 3' end; (4) tagged methylated linker ligation; (5) bisulfite conversion of unmethylated cytosine; (6) enriching a methylation library by PCR; (7) methylation libraries of similar size were mixed and sequenced using the Illumina platform. The invention not only solves the problem of insufficient initial amount of single cell methylation sequencing, but also can carry out large-batch operation processing by introducing the double-label sequence into each single cell sample, thereby being easy to realize automatic flow. The method not only reduces the construction cost of the methylation library of each sample, but also shortens the construction time of the library.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a construction method of a multiple single-cell simplified representative methylation library based on an Illumina sequencing platform.
Background
DNA methylation (DNA methylation) is a form of chemical modification of DNA that alters genetic expression without altering the DNA sequence. DNA methylation refers to the covalent bonding of a methyl group to the cytosine 5' carbon of a genomic CpG dinucleotide under the action of DNA methyltransferase. Numerous studies have shown that DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression.
CpG exists in two forms in mammals: one is dispersed in the DNA sequence; the other exhibits a highly aggregated state, known as CpG islands (CpG island). In normal tissues, 70-90% of scattered CpG is methyl-modified, whereas CpG islands of about 100bp and rich in CpG dinucleotides tend to be unmethylated. CpG dinucleotides are distributed very heterogeneously in the human genome, whereas in certain segments of the genome, CpG remains at or above normal probability. The CpG island is mainly located in a promoter (promoter) and a first exon region of a gene, and about 60% or more of the promoters of the gene contain the CpG island. The GC content of the CpG island is more than 50 percent, and the length is 500-1000 bp. CpG islands are often located near the transcriptional regulatory region of a gene and are associated with 56% of the genes encoded by the human genome. The draft analysis result of the human genome sequence shows that the CpG islands of the human genome are about 28890, 5-15 CpG islands exist in most chromosomes per 1Mb, the average value is that each Mb contains 10.5 CpG islands, and the number of the CpG islands has a good corresponding relation with the gene density. Therefore, the research on the methylation state of CpG islands in the transcribed region of the gene is very important.
Bisulfite treatment of genomic DNA combined with high throughput sequencing remains the gold standard for DNA methylation analysis across the entire genome. In recent years, several single cell-based bisulfite sequencing methods have been reported. The principles of these methods are either based on RRBS or rely on the bisulfite post-treatment linker labeling technique (PBAT) and are not suitable for large-scale single cell methylation sequencing. The harsh conditions of sodium bisulfite treatment can lead to random fragmentation of the DNA, which can lead to substantial loss of DNA if the library DNA is provided with a sequencing linker prior to bisulfite treatment. The multiple single cell simplified representative methylation sequencing process of the invention simplifies the preparation process of the single cell methylation library, so that the single cell methylation library is suitable for large-scale sample processing, and simultaneously reduces the reagent cost and the labor cost. In addition, genome coverage can be directly increased by adding another methylation insensitive enzyme (e.g., HaeIII). Multiple single-cell simplified representative methylation libraries can be manipulated using multichannel pipettors, and library preparation can be easily converted into an automated banking procedure.
Disclosure of Invention
The invention aims to provide a construction method of a multiple single-cell simplified representative methylation library based on an Illumina sequencing platform aiming at the defects of the prior art, and is a method for constructing the multiple simplified representative methylation library by using genomic DNA (deoxyribonucleic acid) which is suitable for large-scale single-cell, multi-cell or sub-nanogram-level purification. Not only reduces the library construction cost of each sample, but also shortens the construction time of the multiple single-cell simplified representative methylation library. By separately sequencing libraries of different fragment sizes, the coverage of CpG islands is greatly improved.
In order to solve the technical problems, the invention adopts the following technical scheme:
a construction method of a multiple single-cell simplified representative methylation library based on an Illumina sequencing platform comprises the following steps:
(1) cell lysis, releasing genomic DNA;
(2) digesting genomic DNA by using restriction endonuclease;
(3) DNA end repair and 3 'end adding A base (i.e. A-Tailing, will dAMP will be incorporated into the blunt end of the 3' end of the DNA fragment);
(4) connecting methylated joints and mixing different label samples;
(5) bisulfite conversion of methylation libraries;
(6) methylation library PCR enrichment (i.e., multiplex single cell simplified representative methylation library PCR enrichment);
(7) methylation libraries of similar size were mixed and sequenced using the Illumina platform.
In one embodiment of the invention, the sample is a library construct from sorted single cells as starting material for multiplex simplified representative methylation sequencing. In addition, in step (1), a purified DNA sample of a multicellular or sub-nanogram grade may be used.
In step (1), the starting DNA for library construction is not limited to single cells, but may be purified DNA samples of multicellular or sub-nanogram grades. Namely, the cells are purified DNA samples of single cell, multi-cell or sub-nanogram grade.
In the step (1), the cell lysis adopts a cell lysis solution, and the cell lysis solution comprises Tris-HCl, EDTA (ethylene diamine tetraacetic acid), NaCl and SDS (sodium dodecyl sulfate), wherein the concentration of Tris-HCl in the cell lysis solution is 10mM, the concentration of EDTA in the cell lysis solution is 10mM, the concentration of NaCl in the cell lysis solution is 10mM, and the mass concentration of SDS in the cell lysis solution is 0.001 g/L, namely the components of the cell lysis solution are 10mM Tris-HCl (pH 7.0), 10mM EDTA, 10mM NaCl and 0.1% (wt/vol) SDS.
Further, the cell lysate can be stored at room temperature (RT, 20-25 ℃) for at least 6 months.
Preferably, proteinase K is added before use.
All the enzyme catalytic reactions in the steps (2), (3) and (4) share the same buffer system, so that the failure of library construction caused by single cell or trace DNA loss due to each purification is avoided. All enzymatic reactions were carried out in the same buffer system. The buffer system adopts a buffer system which is CutSmart buffer.
In the step (2), the CpG island of the genomic DNA is digested by the restriction endonuclease, and the restriction endonuclease can be Msp I, or HaeIII or one, two or more combinations of other corresponding restriction endonucleases.
In the step (3), the DNA end repair and the 3 ' end addition A enzyme are Klenow Exo-enzyme with modified functions, and the enzyme lacks 3 ' -5 ' exonuclease activity.
Preferably, the final concentration of the enzyme in the reaction system is 2.5U.
In the step (4), the DNA ligase is T4 DNA ligase.
Preferably, the final concentration of the enzyme in the reaction system is 100U.
In the step (4), the adaptor sequence connected with the methylated adaptor is a Y-shaped double-stranded structure formed by annealing two single-stranded nucleic acids.
Preferably, the conditions for annealing the two single-stranded nucleic acids to form the Y-type double-stranded structure are that the water bath is carried out at 98 ℃ for 5 minutes, then the water bath is closed, and the temperature is naturally cooled to 25 ℃ at room temperature.
Preferably, the Y-linker is diluted to 0.15. mu.M before use and stored at-80 ℃.
Preferably, all cytosine nucleotides in both single-stranded nucleic acids are methylated; and (3) carrying out phosphorylation treatment on the 5' end of the bottom nucleic acid chain of the Y-shaped double-stranded structure formed after the two single-stranded nucleic acids are annealed.
Preferably, the 3' end of the Y-type duplex is a barcode tag sequence of 6 nucleotides.
Further, after the tag sequence adaptor is added to the sample, the samples with different tags are transferred to the same tube for mixing and magnetic bead purification.
Preferably, 2. mu.l of carrier DNA is added to each tube before purification.
Further, the samples were purified using 1.8x diluted Agencourt AMPure XP magnetic beads.
Preferably, the Agencour AMPure XP magnetic beads need to be diluted with 4 volumes of buffer with 10% PEG/1M NaCl, 0.5% Triton X1-00.
The bisulphite conversion kit used in step (5) is the QiaGen EpiTect fast bisulfitecturon kit.
Preferably, the bisulfite converted DNA library is purified immediately after the temperature has dropped to 20 ℃.
Preferably, the purified DNA after bisulfite conversion should be immediately subjected to polymerase chain reaction amplification or stored at-80 ℃. Specifically, the methylation library should be either immediately subjected to step (6) or stored at-80 ℃ after bisulfite conversion.
In step (6), primers with different tag sequences and DNA polymerase are used to enrich the bisulfite converted library. .
Preferably, the enzyme used for library amplification is 2x KAPA HiFi HotStart Uracil+ReadyMix。
Preferably, the PCR upstream and downstream primers for library amplification are diluted to 25. mu.M with TE buffer and can be stored at-20 ℃ for 1 year
Preferably, 1.5. mu.l of the bisulfite converted library is taken for PCR cycle number optimization before enrichment of the library, ensuring adequate library DNA generation while avoiding the introduction of higher amplification. The reaction system and PCR procedure are as follows:
reaction system:
| Content | Volume(μl) | Final concentration |
| BS converted DNA | 1.5 | |
| 2x KAPAHiFi HotStart Uracil+ReadyMix | 25 | 1x |
| Primer mix(25μM for each) | 1 | 0.5μM for each |
| H2O | 22.5 | |
| |
50 |
PCR procedure:
preferably, we recommend starting at 10 PCR cycles and successively incrementing by 2 PCR cycles to establish the optimal number of cycles.
And (7) purifying the library after PCR enrichment and mixing the libraries with different fragment sizes.
Further, the amplified library was purified using 1.3X diluted Agencourt AMPure XP magnetic beads.
Preferably, the Agencour AMPure XP magnetic beads need to be diluted with 3 volumes of buffer with 10% PEG/1M NaCl and 0.5% Triton X1-00.
Further, the purified library is subjected to gel cutting and recovery.
Preferably, gel electrophoresis and gel recovery are performed using 3% NuSieve 3:1agarose TBE-gel
Preferably, the DNA fragments with bands of 600bp at 170-and 1,000bp at 600-are recovered by cutting gel for each mixed sample.
Preferably, the library recovered from the cut gel is purified using the QiaGen MinElute gel purification kit.
Further, quality control and mixing are carried out on the samples recovered by the cutting glue.
Preferably, the Agilent high sensitive chip is used for quality control of library size and concentration.
Preferably, libraries of the same band size are mixed in equal proportional molar amounts depending on how much the data amount is partitioned.
Further, the pooled library was subjected to on-machine sequencing.
Preferably, sequencing loading should be clustered at 60% density of a conventional Illumina library.
Preferably, the mixed sequencing library should incorporate 30% -50% of the phiX equilibrium library to ensure library diversity.
Compared with the prior art, the invention has the following advantages:
1. the construction method of the multiple single cell simplified representative methylation library can be used for constructing the methylation library by utilizing single cell, multiple cell or sub-nanogram purified genome DNA in a large scale. The whole process of generating 96 multiple single-cell simplified representative methylation libraries only needs 3 days, and the construction time of the whole methylation library is greatly saved.
2. The independently developed construction method of the multiple single-cell simplified representative methylation library can remarkably improve the coverage of CpG islands, including promoters and enhancers, and provides DNA methylation information in an economic and efficient manner; and allows analysis of DNA methylation information of hundreds of single cells at a time.
3. The construction method of the multiple single-cell simplified representative methylation library utilizes a method that a sample set can mix 24 single-cell methylation libraries and Agencour AMPure XP magnetic beads to remove linker dimers, eliminates the complexity of the single-cell methylation libraries and the loss caused by electrophoresis selection, allows large-scale library generation, is easy to automatically set, and reduces the total cost of each sample.
4. The construction method of the multiple single-cell simplified representative methylation library can use ultra-low content of DNA and single cells as starting materials for research, and is more suitable for researching DNA methylation conditions of single cells in normal development and human diseases (particularly cancers) at different periods.
5. The construction method of the multiple single-cell simplified representative methylation library can simultaneously research the DNA methylation states of thousands of cells at relatively low cost, which opens a new door for the dissection of cell circuits and the exploration of epigenetic basis for disease progression.
6. The construction technology of the multiple single-cell simplified representative methylation library can better understand the characteristics of cancer tumor cells in clinic, and the tumor cells actively respond to drug treatment so as to drive the clonal evolution process and further obtain the overall clinical result.
Drawings
FIG. 1 is a schematic flow chart of a method for constructing a simplified representative methylation library of multiple single cells based on an Illumina sequencing platform according to the present invention;
FIG. 2 is a schematic diagram of a 96 single cell library construction scheme;
FIG. 3 is a gel image and a combinatorial electrophoresis image of a simplified representative methylation library of 4 groups of 24 single cells generated using an Agilent bioanalyzer high sensitivity DNA analysis kit;
FIG. 4 is a quality control diagram of a simplified representative methylation library of single cells recovered from 170-400bp and 400-1,000bp using an Agilent bioanalyzer high sensitivity DNA analysis kit;
FIG. 5 is a gel electrophoresis image of PCR products after library enrichment;
Detailed Description
The inventive library of single-cell simplified representative bisulfite sequencing is constructed after the specific linker containing the tag is added, and is directly mixed into a sample for PCR enrichment and downstream sequencing. Greatly optimizes the operation flow and reduces the economic cost and the labor cost. Meanwhile, the problem of library establishment failure caused by insufficient initial amount of single cells is solved, and the method is an innovation of the traditional single cell methylation sequencing technology.
In order to enable researchers and clinical staff in the related art to better understand the scheme of the invention, the following detailed description is provided in combination with the embodiment mode and the attached drawings. However, the present invention is not limited to the following embodiments.
The flow schematic diagram of the construction method of the multiple single-cell simplified representative methylation library based on the Illumina sequencing platform is shown in figure 1; the scheme of the construction process of 96 single-cell libraries is shown in FIG. 2; gel images and combined electropherograms of 4 sets of 24 single-cell simplified representative methylation libraries generated using an agilent bioanalyzer high sensitivity DNA analysis kit are shown in fig. 3; the quality control graph of the single cell simplified representative methylation library recovered by 170-400bp and 400-1,000bp using the Agilent bioanalyzer high sensitivity DNA analysis kit is shown in FIG. 4.
EXAMPLE 1 Single cell lysis
1. Mu.l of 0.1 XCutSmart buffer was dispensed to each well in a 96-well PCR plate. The CutSmart buffer, i.e., the cell lysate component, was 10mM Tris-HCl (PH 7.0), 10mM EDTA, 10mM NaCl, and 0.1% (wt/vol) SDS.
2. One single cell was sorted in each well of a 96-well plate. If the sorted cells are not ready for use in making a single-cell reduced representative methylation library, the 96-well plate can be sealed with a polymerase chain reaction seal "F" membrane (BIORAD, CAT # MSF1001) and stored in a-80 ℃ freezer until further processing.
3. A mixture of cell lysis buffers was prepared according to the following table.
Note that more than 20% of the master batch mixture was additionally prepared to compensate for the loss of the reagent during pipetting.
4. 19. mu.l of the above buffer was dispensed to each well of row A of 12 wells of a 96-well PCR plate, the 96-well plate was sealed and briefly centrifuged, and then 2. mu.l of the master mix was transferred to each well of the sample plate using a 12-channel pipette. Note that: the mixed sample is not pipetted up and down because the cells may stick to the tip of the tip.
5. The sample plate was sealed and then briefly centrifuged.
The critical step is to ensure that each sample well is completely sealed. If the sealing is not effective, it may result in loss of the sample during the cell lysis step.
6. Gently shaking the sample plate to mix the cell lysate well in each sample; followed by brief centrifugation to centrifuge any liquid accumulated in the cover membrane to the bottom of the tube.
Note that the sample plate was gently placed into a special pan mixer for mixing.
7. The sample plate was placed in a 96-well thermal cycler that was preprogrammed as follows:
| Program | Temperature(℃) | Duration(Minutes) |
| 1 | 45 | 120 |
| 2 | 75 | 30 |
| 3 | 4 | hold |
8. the 96-well sample plate was centrifuged at 3000 rpm for 30 seconds and the cell lysis step was completed.
Example 2 restriction enzyme digestion
1. The Msp i digestion reaction mixture was prepared as follows:
note that: another methylation insensitive restriction enzyme may be added to increase coverage of CpG islands. Ensure that different restriction endonucleases can be compatible in the same CutSmart buffer.
2. A19. mu.l aliquot of the Msp I digestion reaction mixture was dispensed into each of the 12 wells in row C of a 96-well PCR plate.
3. The 96-well sample plate cover film was carefully removed to avoid cross-contamination of the samples, and then 2 μ Ι of digestion mixture was transferred to each sample well using a 12-channel pipette. The 96-well plate was briefly centrifuged.
4. The reaction was mixed by gentle shaking and centrifuged briefly.
5. The sample plate was placed in a thermal cycler with the thermal lid temperature preset to 80 ℃. The incubation reactions were performed according to the following procedure:
| Program | Temperature(℃) | Duration(Minutes) |
| 1 | 37 | 30 |
| 2 | 70 | 10 |
| 3 | 4 | hold |
note that: the thermal lid temperature of most thermal cyclers is set to 105 ℃. The temperature of the lid was lowered to avoid the reaction system changing during the long incubation period.
6. After the incubation was complete, the sample plate was centrifuged at 3000 rpm for 30 seconds.
Example 3 end repair
1. The formulation of the end repair system was carried out according to the following formulation:
2. 19. mu.l of the above reaction was dispensed to each of 12 wells in row E of a 96-well PCR plate.
3. The 96-well sample plate cover film was carefully removed to avoid cross-contamination of the samples, and then 2 μ Ι of the end repair mixture was transferred to each sample well using a 12-channel pipette. The 96-well plate was briefly centrifuged.
4. The reaction was mixed by gentle shaking and centrifuged briefly.
5. The sample plate was placed in a thermal cycler with the thermal lid temperature preset to 80 ℃. The incubation reactions were performed according to the following procedure:
| Program | Temperature(℃) | Duration(Minutes) |
| 1 | 30 | 10 |
| 2 | 37 | 10 |
| 3 | 70 | 5 |
| 4 | 4 | Hold |
6. after the incubation was complete, the sample plate was centrifuged at 3000 rpm for 30 seconds.
EXAMPLE 4 Joint attachment
1. Prepare the reaction system for linker attachment as follows:
single cell simplified representative methylated linker after synthesis, methylated linkers were dissolved to 200. mu.M using low TE buffer as stock solution, part of the stock solution was diluted to 0.15. mu.M working solution, aliquoted to 20. mu.L in 96-well PCR plates, and the linkers were stored at-80 ℃ after blocking.
2. 14 μ l of the ligation reaction mixture was dispensed to each of the 12 wells in row G of the 96-well PCR plate.
3. Carefully remove the 96-well sample plate cover film to avoid cross-contamination of the samples, and then transfer 1.5 μ Ι of adaptor ligation mix into each sample well using a 12-channel pipette. The 96-well plate was briefly centrifuged.
4. The reaction was mixed by gentle shaking and centrifuged briefly.
5. 1.5 μ l of diluted, barcode-tagged methylated linker was added to each sample well using a 12-channel pipette.
Note that: the adapter solution may appear under the cover membrane after thawing. Before use, the adapter solution was centrifuged at 3000 rpm for 1 minute to collect the adapter at the bottom of the tube. Care should be taken when tearing the sealing film to prevent cross-contamination of the joints.
After the 6.96 well sample plate was sealed, the reaction system was mixed by gentle shaking and briefly centrifuged.
7. The ligation reaction was incubated at 16 ℃ for 1-3h, lid temperature was set to 25 ℃ and ligase was inactivated at 70 ℃ for 15 min.
8. After the incubation was complete, the sample plate was centrifuged at 3000 rpm for 1 minute.
9. The 24 different barcode-labeled samples were placed in the same 1.5ml Eppendorf L oBind centrifuge tube, then the 24 sample wells were washed with 30 μ l of low TE buffer and finally mixed with the sample.
Note that each set of different barcode-labeled samples was pooled in the same 1.5ml Eppendorf L oBind centrifuge tube using the same tip, after which 24 sample wells were washed thoroughly with 30. mu.l of low TE buffer using a new tip transfer and finally mixed with the sample.
10. Mu.l of carrier DNA (50 ng/. mu.l of fragmented, dephosphorylated bacterial DNA) was added to each set of mixed samples with a pipette.
11. To each mixed sample library was added a 1.8-fold dilution of Agencour AMPure XP magnetic beads and the tube was rotated at room temperature for 30 minutes to facilitate binding of the magnetic beads to the library DNA.
Note that: the DNA content of each library was low and the diluted beads were sufficient to bind to the library DNA. The AgencourtAMPure XP magnetic beads were diluted with 4 volumes of buffer containing 10% PEG/1M NaCl, 0.5% Triton X1-00.
12. Briefly centrifuge the sample tube, place the sample tube in DynaMagTM-2 (L if Technologies) magnetic beads were separated on a magnetic rack, separated at room temperature for 10 minutes, and the supernatant solution was carefully removed, taking care not to touch the beads.
13. The beads were washed twice with 1 ml of 80% freshly prepared ethanol.
14. After the ethanol was completely evaporated, the library DNA was eluted with 40. mu.l of low TE buffer.
The key step is that it takes a little time to dry the Agencourt AMPure XP beads due to their large size, care is taken not to air the beads too dry, the beads may stick to one side of an Eppendorf L oBind centrifuge tube, and if necessary, they can be scraped off with a pipette tip.
Example 5 bisulfite conversion
1. Methylation processing was performed according to the product instructions of the QIAGEN epitec rapid bisulfite conversion kit with some modifications: we used a two cycle bisulfite protocol, as shown below; and when necessary, the QIAGEN spin column was replaced with a Zymo spin column (Cat #, C1003).
The key steps are as follows: bisulfite converted DNA was immediately cleared when the temperature dropped to 20 ℃.
2. Bisulfite converted DNA was eluted with 30. mu.l of EB buffer.
The key steps are as follows: the bisulfite converted DNA should be immediately amplified by polymerase chain reaction or stored at-80 ℃.
Note that: if left at room temperature or 4 ℃, bisulfite converted deoxyribonucleic acid will degrade rapidly, causing library construction to fail.
Example 6 library enrichment
(optionally) if there are multiple similar pooled libraries available, it is recommended to use one of the pooled libraries to validate the PCR cycle and perform the final enrichment of the pool DNA with the lowest number of cycles from which sufficient pool DNA can be generated, as follows:
| Volume(μl) | Final concentration | |
| BS converted DNA | 1.5 | |
| 2x KAPA HiFi HotStart Uracil+ReadyMix | 25 | 1x |
| Primer mix(25μM for each) | 1 | 0.5μM for each |
| H2O | 22.5 | |
| Total | 50 |
2. mu.l of each of the above 50. mu.l PCR reaction mixtures was dispensed into 4 different PCR wells. Simply centrifuging the PCR plate hole, and carrying out subsequent PCR amplification, wherein the amplification parameters are as follows:
note that: typically, starting with 10 cycles of PCR, there are a total of 4 different cycle numbers in 2 cycle increments.
PCR products were run on 4-20% PAGE gels at 130 volts for 1 hour; the gel was prepared at 1: staining in 10000 diluted SYBR green I for 15 min.
4. The gel was scanned and the optimal number of PCR cycles was determined to enrich for library DNA.
5. After determining the optimal number of PCR cycles, the pooled library set was subjected to amplification enrichment.
6. The following PCR reaction system was prepared:
7. mu.l of the reaction mixture was dispensed into 3 wells of a 96-well PCR plate and subjected to PCR amplification according to the following protocol:
| Cycle number | Denature | Anneal | Extend |
| 1 | 98℃,45s | ||
| 6 | 98℃,20s | 58℃,30s | 72℃,1min |
| 12～14 | 98℃,20s | 65℃,30s | 72℃, |
| 1 | 72℃,5min |
stoppable point: the library DNA after PCR amplification can be stored at-20 ℃ for 2-3 weeks. If longer storage times are required, please store the library DNA in a refrigerator at-80 ℃.
Example 7 preparation of sequencing libraries
1. The PCR products of the same pool were transferred to a new Eppendorf tube, purified with 1.3-fold dilutions of AgencourtAmpure XP magnetic beads (1: 3), and incubated with rotation at room temperature for 10min to promote binding of the magnetic beads to the library DNA.
2. After brief centrifugation of the sample tube, at DynaMag TM2 magnetic beads were separated for 10 minutes on a magnetic rack and the supernatant solution was carefully removed without disturbing the beads.
3. The beads were washed twice with 1 ml 80% freshly prepared ethanol.
4. The library DNA was eluted with 20. mu.l of low TE buffer.
5. Preparation of 3% NuSieve 3:1agarose gel.
6. Library DNA was purified in 3% NuSieve 3: electrophoresis on a 1agarose TBE gel for 2.5 hours, at 1: 10000 diluted SYBR green I (deionised H)2Dilution in O) for 1 hour
7. And (5) cutting and recovering target bands according to the double-sided DNA marker in the gel hole (figure 5). The DNA fragments of 170-400bp and 400-1000bp were purified using QIAGENNINELEUTE gel purification kit to remove PCR primer dimers.
8. The final library DNA was eluted with 15. mu.l EB buffer.
9. Library DNA QC the size and molar concentration of the library DNA fragments were determined using an Agilent HS high sensitive chip.
10. Libraries of the same band size were mixed in equal molar amounts depending on the amount of data required.
11. The library mixed by two different fragment sizes is subjected to 100bp double-end sequencing on an Illumina sequencing platform. As with other bisulfite sequencing libraries, single cell reduced representative methylation libraries should also be loaded onto sequencing chips (flow cells) at relatively low cluster densities (-60% of the density of conventional Illumina sequencing libraries). Meanwhile, 30-50% of phiX DNA equilibrium library is added to increase the base diversity of the sequencing library and improve the sequencing quality.
The above is only a specific embodiment of the present invention, but the technical features of the present invention are not limited thereto. Any simple changes, equivalent substitutions or modifications made based on the present invention to solve the same technical problems and achieve the same technical effects are within the scope of the present invention.
Claims (10)
1. A construction method of a multiple single-cell simplified representative methylation library based on an Illumina sequencing platform is characterized by comprising the following steps:
(1) cell lysis, releasing genomic DNA;
(2) digesting genomic DNA by using restriction endonuclease;
(3) DNA end repair and 3' end addition of A;
(4) methylated linker ligation;
(5) bisulfite conversion of methylation libraries;
(6) PCR enrichment of methylation libraries;
(7) methylation libraries with similar fragment sizes were pooled and sequenced using the Illumina platform.
2. The method for constructing the simplified representative methylation library of multiple single cells based on the Illumina sequencing platform according to claim 1, wherein in the step (1), the cells are single cells, multiple cells or purified DNA samples with sub-nanogram grade.
3. The method for constructing the simplified representative methylation library of multiple single cells based on the Illumina sequencing platform according to claim 1, wherein in the step (1), a cell lysate is adopted for cell lysis, and the cell lysate comprises Tris-HCl, EDTA, NaCl and SDS, wherein the concentration of Tris-HCl in the cell lysate is 10mM, the concentration of EDTA in the cell lysate is 10mM, the concentration of NaCl in the cell lysate is 10mM, and the mass concentration of SDS in the cell lysate is 0.001 g/L.
4. The method for constructing the simplified representative methylation library of multiple single cells based on the Illumina sequencing platform according to claim 1, wherein in the step (2), the restriction enzyme is Msp I or HaeIII.
5. The method for constructing the simplified representative methylation library of multiple single cells based on the Illumina sequencing platform according to claim 1, wherein in the step (4), the linker sequence connected with the methylation linker is a Y-shaped double-stranded structure formed by annealing two single-stranded nucleic acids.
6. The method for constructing the simplified representative methylation library of multiple single cells based on the Illumina sequencing platform according to claim 5, wherein all cytosine nucleotides in both single-stranded nucleic acids are methylated; and (3) carrying out phosphorylation treatment on the 5' end of the bottom nucleic acid chain of the Y-shaped double-stranded structure formed after the two single-stranded nucleic acids are annealed.
7. The method for constructing the simplified representative methylation library of multiple single cells based on the Illumina sequencing platform according to claim 6, wherein the 3' end of the Y-type double-stranded structure is a tag sequence with 6 nucleotides.
8. The method for constructing the simplified representative methylation library of the multiple single cells based on the Illumina sequencing platform, according to claim 1, wherein in the step (7), the methylation libraries with bands of 170bp-400bp and 400bp-1000bp are respectively cut and recovered by gel.
9. The method for constructing the Illumina sequencing platform-based multiple single-cell simplified representative methylation library according to claim 1, wherein Illumina platform mixed sequencing is performed in step (7), and the loading amount is generated according to 60% of the Illumina library density.
10. The method for constructing the Illumina sequencing platform-based multiple single-cell simplified representative methylation library according to claim 1, wherein Illumina platform mixed sequencing is performed in step (7), and 30% -50% of phiX equilibrium library should be doped in the mixed sequencing library.
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