CN113463203A - Construction method of in-situ sequencing library for realizing multiple RNA in-situ detection - Google Patents
Construction method of in-situ sequencing library for realizing multiple RNA in-situ detection Download PDFInfo
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Abstract
The invention discloses a method for constructing an in-situ sequencing library for realizing multiple RNA in-situ detection, which constructs the in-situ sequencing library by utilizing a specially designed nucleic acid probe technology and realizes the highly multiple RNA in-situ detection by utilizing the in-situ sequencing technology and a fluorescence microscopic imaging technology; the complete tag sequence is placed on the two chains of the identification probe pair, so that compared with the situation that the complete tag sequence is placed at one end, the average distance between a sequencing position and a connecting position is shortened, and the signal intensity and specificity are improved.
Description
Technical Field
The invention belongs to the technical field of RNA in-situ expression analysis, and particularly relates to a construction method of an in-situ sequencing library for realizing multiple RNA in-situ detection.
Background
Spatial transcriptomics, which is an emerging gene expression analysis technique in recent years, refers to a series of techniques that enable spatially localized and quantitative analysis of genes. Current spatial transcriptomics methods are mainly classified into two major categories, microscopic imaging-based methods and sequencing-based methods. The former is an in situ sequencing technology and a coding technology based on single molecule fluorescence in situ hybridization, and the latter is a series of primer coding addressing methods based on microarray.
The in situ sequencing technology is a space transcriptomics technology for realizing in situ analysis of multiple gene expressions depending on a new generation sequencing chemical technology, and can be divided into full transcriptome in situ sequencing and targeted in situ sequencing, wherein the latter technology needs to use a probe to construct an in situ sequencing library. This technique is mainly used to detect different genes by performing multiple rounds of sequencing of signals from a single RNA molecule in a cell or tissue, resulting in a string of signals encoded by different color fluorescence. Spatial transcriptomics technologies represented by in situ sequencing mainly include in situ sequencing technology, fluorescence in situ sequencing technology (FISSSEQ) and STARmap (spatial-resolved transcription amplification read out mapping) technology. The FISEBS does not use a probe to carry out targeted detection, but directly carries out reverse transcription of RNA into cDNA in situ by adopting a method similar to a next generation sequencing technology, then carries out cyclic amplification, and directly carries out in situ sequencing chemistry of a longer segment in a cell, thereby obtaining a series of gene sequence segments to realize multiple RNA in situ detection. In situ sequencing and STARmap technology are based on capture of target short sequences by padlock probes or coding of different genes by using tagged padlock probes so as to realize simultaneous detection, and sequencing by the technology has relatively limited target genes which can be detected simultaneously due to short sequencing length (4-6nt) (4N-order species tags).
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for constructing an in-situ sequencing library for realizing in-situ detection of multiple RNAs.
The technical scheme of the invention is as follows:
a method for constructing an in-situ sequencing library for realizing in-situ detection of multiple RNAs comprises the following steps:
(1) designing at least one recognition probe pair according to a target sequence on the target RNA; each recognition probe pair consists of an upstream recognition probe and a downstream recognition probe, wherein the upstream recognition probe contains an upstream label sequence, the downstream recognition probe contains a downstream label sequence, and the upstream label sequence and the downstream label sequence form a complete label sequence for reading the corresponding target sequence;
(2) delivering the at least one recognition probe pair into the cell, so that the recognition probe pair is recognized and hybridized with the target sequence on the target RNA, wherein the 3 'end of the upstream recognition probe and the 5' end of the downstream recognition probe are close to each other and are connected through DNA ligase to form at least one chain DNA molecule;
(3) hybridizing at least one chain DNA molecule obtained in the step (2) with a DNA splint sequence to enable the 5 'end and the 3' end of the at least one chain DNA molecule to be close to each other, and then connecting the at least one chain DNA molecule and the DNA splint sequence through DNA ligase to form at least one circular DNA molecule;
(4) and performing rolling circle amplification by taking the at least one annular DNA molecule as a template to obtain amplification products, namely the in-situ sequencing library, decoding different labels in the in-situ sequencing library by using an in-situ sequencing technology to obtain a complete label sequence on each amplification product, and further obtaining the types of target RNAs detected by different amplification products, so that in-situ expression information of different target RNAs is obtained, and the in-situ detection of the multiple RNAs is realized.
In a preferred embodiment of the present invention, the target RNA includes RNA of the cell itself and RNA in which an exogenous gene is expressed inside the cell.
In a preferred embodiment of the invention, the DNA ligase is Splint R ligase.
In a preferred embodiment of the invention, the cells are free cells to be tested or cells within a tissue.
In a preferred embodiment of the present invention, the target RNA includes RNA of the cell itself and RNA in which an exogenous gene is expressed inside the cell, which is a free cell to be tested or a cell in a tissue.
In a preferred embodiment of the present invention, the target RNA includes RNA of the cell itself and RNA in which an exogenous gene is expressed inside the cell, and the DNA ligase is Splint R ligase.
In a preferred embodiment of the invention, the cell is an isolated test cell or a cell within a tissue and the DNA ligase is Splint R ligase.
In a preferred embodiment of the present invention, the target RNA includes RNA of the cell itself and RNA in which an exogenous gene is expressed inside the cell, the cell is an isolated cell to be tested or a cell in a tissue, and the DNA ligase is Splint R ligase.
The invention has the beneficial effects that:
1. the invention constructs an in-situ sequencing library by utilizing a specially designed nucleic acid probe technology, and realizes highly multiplex RNA in-situ detection by utilizing the in-situ sequencing technology and the fluorescence microscopic imaging technology.
2. According to the invention, the complete label sequence is placed on the two chains of the identification probe pair, compared with the situation that the complete label sequence is placed at one end, the average distance between the sequencing position and the connection position is shortened, and the signal intensity and specificity are improved.
3. In the invention, the complete label sequence is placed on the two strands of the identification probe pair, and compared with the situation that the complete label is placed at one end, if the probe pair is mismatched, the generated error label can be eliminated.
Drawings
Fig. 1 is an experimental schematic diagram of embodiment 1 of the present invention.
FIG. 2 is a schematic diagram of in situ sequencing library generated in example 1 of the present invention decoded by in situ sequencing.
FIG. 3 is a graph showing the results of the experiment in example 1 of the present invention.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
Example 1 method for constructing gene expression in situ analysis library by in situ detecting 30 mRNA for experimental samples by paraffin tissue section of colorectal cancer
The experimental principle of this example is shown in fig. 1, and the specific steps are as follows:
firstly, paraffin tissue pretreatment:
baking paraffin tissue slices in an oven at 65 deg.C for 30min, immediately soaking in a staining jar containing xylene for 10min, and replacing xylene and soaking for 10 min. And taking out the glass slide from the second staining jar containing the dimethylbenzene, immediately putting the glass slide into the staining jar containing the absolute ethyl alcohol, soaking for 2min, replacing the absolute ethyl alcohol, and soaking for 2 min. Taking out, immediately placing the glass slide into a staining jar containing 95% ethanol, soaking for 2min, replacing 95% ethanol, and soaking for 2 min. Taking out, immediately placing the glass slide into a staining jar containing 85% ethanol, soaking for 2min, replacing 85% ethanol, and soaking for 2 min. Taking out, immediately placing the glass slide into a staining jar containing 70% ethanol, soaking for 2min, replacing 70% ethanol, and soaking for 2 min.
Taking out the glass slide from the ethanol solution, and soaking the glass slide in DEPC-H2Soaking in DEPC-PBS for 2min after 5min in O, and washing off residual ethanol. 4% PFA in DEPC-PBS was dropped onto the slide, completely covering the tissue sample, incubated at room temperature for 10min and washed away with DEPC-PBS. 30mL of 0.1M HCl was placed in a staining jar and preheated to 37 ℃, 30uL of 0.1mg/mL pepsin was added, the mixture was mixed well and immediately the slide was immersed in the mixture and incubated at 37 ℃ for 30 min. Taking out the glass slide and soaking the glass slide in DEPC-H2Soaking in O for 5min, and soaking in DEPC-PBS for 2 min. Respectively passing through gradient ethanol: dehydrating at 70%, 85% and 100% for 2min respectively, and air drying. Finally, the mixture is washed three times by DEPC-PBS-Tween20 for later use.
(II) in-situ nucleic acid detection:
(1) double ligation probe hybridization:
50 μ L of hybridization mixture containing final concentrations of 6 XSSC, 10% formamide and 0.1 μ M of each of the double-linked probes was dropped onto the slide and incubated at 37 ℃ for 4h to allow each of the recognition probe pairs to hybridize sufficiently with the target sequence of the corresponding gene.
The ligation probe sequences are shown in the following table:
(2) the above identification talks about the first step of connection:
after washing three times with DEPC-PBS-Tween20, 50. mu.L of ligation mixture containing 25% glycerol, 0.2. mu.g/. mu.L BSA, 1 XSSplintR buffer (NEB), 0.1U/. mu.L SplintR ligand (NEB) and 1U/. mu.L RiboLock RNase inhibitor (Thermo) was added to the sample and incubated at 37 ℃ for 30 min.
(3) Hybridization of splint primers:
after three washes with DEPC-PBS-Tween20, 50 μ L of hybridization mix containing final concentrations of 6 XSSC, 10% formamide, and 0.5 μ M splint primers was added to the samples and incubated at 30 ℃ for 30 min.
The splint primer sequence is as follows: 5'-ggctccactaaatagacgca-3', SEQ ID NO. 61.
(4) Probe cyclization and rolling circle amplification:
after washing three times with DEPC-PBS-Tween20, 50. mu.L of a hybridization mixture containing 5% glycerol, 0.2. mu.g/. mu.L BSA, 1 XPhi 29 polymerase buffer (Thermo), 1mM dNTPs, 0.1U/. mu. L T4 DNA ligase (Thermo) and 1U/. mu.L Phi29 DNA polymerase (Thermo) was added to the sample and incubated at 37 ℃ for 6h or at 30 ℃ overnight.
(5) Detection probe hybridization and image acquisition
Hybridizing the rolling circle amplification product with a detection probe (5'-tgcgtctatttagtggagcc-3', SEQ ID NO.62, and Cy3 marked at the 5 ' end), and finally, carrying out cell nucleus staining by using DAPI for detection, wherein the detection is carried out specifically: samples were washed three times with DEPC-PBS-Tween20, added to the reaction mixture containing 0.1 μ M detection probe, 20% formamide and 2 xssc, incubated at 37 ℃ for 30min, and then subjected to gradient ethanol: 70%, 85%, 100% dehydration treatment, each for 2 min. And (5) air drying. Finally, the mixture was added with 0.5. mu.g/mL DAPI
Gold antipade mount (Fermantas) was incubated at room temperature for 10min before fluorescent microscopy and photographed.
(6) Elution of detection probes
And (3) putting the photographed and imaged glass slide into 70% ethanol for incubation, and washing away the cover glass and the mounting agent. Then, elution buffer was added to the reaction zone at a final concentration of 60% formamide and 0.1% Triton-X, and the mixture was incubated at 37 ℃ for 10min, and the above steps were repeated 3 times. The samples were finally washed three times with DEPC-PBS-Tween 20.
The specific principle of the (third) four-round in situ nucleic acid detection is shown in fig. 2, and specifically comprises the following steps: :
(1) anchor primer hybridization and fluorescent probe ligation
To the sample was added 50. mu.L of a sequencing reaction mixture containing the anchor primer at a final concentration of 0.2. mu.M, each fluorescent probe at 0.2. mu.M, 1 XT 4 DNA ligase buffer, 1mM ATP and 0.1 u/. mu. L T4 DNA ligase, and the mixture was incubated at 30 ℃ for 1 hour. After washing three times with DEPC-PBS-Tween20, the air was dried. Finally, Gold antipide mount (Fermantas) containing 0.5. mu.g/mL DAPI was added and incubated at room temperature for 10min before fluorescence microscopy and photography.
(2) Elution of fluorescent probes
And (3) putting the photographed and imaged glass slide into 70% ethanol for incubation, and washing away the cover glass and the mounting agent. Then, elution buffer was added to the reaction zone at a final concentration of 60% formamide and 0.1% Triton-X, and the mixture was incubated at 37 ℃ for 10min, and the above steps were repeated 3 times. The samples were finally washed three times with DEPC-PBS-Tween 20.
(3) Repeating the steps (1) and (2) three more times, wherein the sequences of the anchor primer and the fluorescent probe are shown in the following table:
| serial number | Name (R) | Sequence of | 5' labelling | 3' labelling |
| SEQ ID NO.63 | Anchor primer-L | tgcgtctatttagtg | P | / |
| SEQ ID NO.64 | Anchor primer-R | ctatttagtggagcc | / | / |
| SEQ ID NO.65 | Seq-base1-A | ctatcnnan | Cy5 | / |
| SEQ ID NO.66 | Seq-base1-T | ctatcnntn | AF488 | / |
| SEQ ID NO.67 | Seq-base1-C | ctatcnncn | TXR | / |
| SEQ ID NO.68 | Seq-base1-G | ctatcnngn | Cy3 | / |
| SEQ ID NO.69 | Seq-base2-A | ctatcnnna | Cy5 | / |
| SEQ ID NO.70 | Seq-base2-T | ctatcnnnt | AF488 | / |
| SEQ ID NO.71 | Seq-base2-C | ctatcnnnc | TXR | / |
| SEQ ID NO.72 | Seq-base2-G | ctatcnnng | Cy3 | / |
| SEQ ID NO.73 | Seq-base3-A | annnctatc | P | Cy5 |
| SEQ ID NO.74 | Seq-base3-T | tnnnctatc | P | AF488 |
| SEQ ID NO.75 | Seq-base3-C | cnnnctatc | P | TXR |
| SEQ ID NO.76 | Seq-base3-G | gnnnctatc | P | Cy3 |
| SEQ ID NO.77 | Seq-base4-A | nannctatc | P | Cy5 |
| SEQ ID NO.78 | Seq-base4-T | ntnnctatc | P | AF488 |
| SEQ ID NO.79 | Seq-base4-C | ncnnctatc | P | TXR |
| SEQ ID NO.80 | Seq-base4-G | ngnnctatc | P | Cy3 |
The sequence n is any one of a, t, c and g.
(4) Analysis of Gene
By synthesizing the signals of the four rounds of sequencing, each signal point obtains the color sequence of the four rounds, and the base sequence on the probe designed before can know the signal of which gene the point is, as shown in FIG. 3.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
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<213> Artificial Sequence (Artificial Sequence)
<400> 27
ctcttgccta cgccacttcc tatctccttg cgtctatt 38
<210> 28
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
tagtggagcc acctctatcc tttgccttga cgatacag 38
<210> 29
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
tttgccatcc actatcttcc tatctcattg cgtctatt 38
<210> 30
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
tagtggagcc tcctctatcc tttggtctca gacaccac 38
<210> 31
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 31
ttgccatcca ctacttttcc tatcacagtg cgtctatt 38
<210> 32
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 32
tagtggagcc gcctctatcc ttttcagatg acacgacc 38
<210> 33
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 33
ttgggcagga cgtcagttcc tatcacgctg cgtctatt 38
<210> 34
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 34
tagtggagcc ggctctatcc tttgccgcag ctgttcac 38
<210> 35
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 35
aaagtcaaag aggtgctttc ctatctcagt gcgtctatt 39
<210> 36
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 36
tagtggagcc atctctatcc ttgccgagag gcgatgggc 39
<210> 37
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 37
aatattggag aggccttcct atctcactgc gtctatt 37
<210> 38
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 38
tagtggagcc tgctctatcc ttgatcctgg cctgaact 38
<210> 39
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 39
tcgaaggtga catagtgttc ctatctcgtt gcgtctatt 39
<210> 40
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 40
tagtggagcc cgctctatcc ttgctgtagt agagtccg 38
<210> 41
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 41
attctcctcg gtgtccttcc tatctctttg cgtctatt 38
<210> 42
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 42
tagtggagcc cactctatcc ttggtgttcg cctcttgac 39
<210> 43
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 43
gaatcactgc cagtcattcc tatctcgatg cgtctatt 38
<210> 44
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 44
tagtggagcc tcctctatcc ttggattttt aagaaaaa 38
<210> 45
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 45
agttctcgaa gtctgacttc ctatctcact gcgtctatt 39
<210> 46
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 46
tagtggagcc gcctctatcc ttgctccaaa ttccctgg 38
<210> 47
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 47
ttcttgctct atggtcgttc ctatctcagt gcgtctatt 39
<210> 48
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 48
tagtggagcc acctctatcc ttagttagca gaatcttga 39
<210> 49
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 49
cacactcaca ctcatattcg atagtcggtg cgtctatt 38
<210> 50
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 50
tagtggagcc agctctatcc ttccaacttc catgcaca 38
<210> 51
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 51
gatctccacg tagtccttcc tatctcgctg cgtctatt 38
<210> 52
<211> 37
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 52
tagtggagcc aactctatcc ttgtatttct ccccgtt 37
<210> 53
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 53
atgctggttg tacaggttcc tatctcgttg cgtctatt 38
<210> 54
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 54
tagtggagcc aactctatcc ttccgaggcg cccgggtt 38
<210> 55
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 55
cttaacaggt gctttggttc ctatctcact gcgtctatt 39
<210> 56
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 56
tagtggagcc agctctatcc ttacttgggg gtcaggagt 39
<210> 57
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 57
actccctgtc ctgaatttcc tatctcagtg cgtctatt 38
<210> 58
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 58
tagtggagcc agctctatcc tttctttgca gttggtca 38
<210> 59
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 59
atgtactcga tctcatcttc ctatctcact gcgtctatt 39
<210> 60
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 60
tagtggagcc ccctctatcc ttacaggatg gcttgaag 38
<210> 61
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 61
ggctccacta aatagacgca 20
<210> 62
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 62
tgcgtctatt tagtggagcc 20
<210> 63
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 63
tgcgtctatt tagtg 15
<210> 64
<211> 15
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 64
ctatttagtg gagcc 15
<210> 65
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 65
ctatcnnan 9
<210> 66
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 66
ctatcnntn 9
<210> 67
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 67
ctatcnncn 9
<210> 68
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 68
ctatcnngn 9
<210> 69
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 69
ctatcnnna 9
<210> 70
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 70
ctatcnnnt 9
<210> 71
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 71
ctatcnnnc 9
<210> 72
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 72
ctatcnnng 9
<210> 73
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 73
annnctatc 9
<210> 74
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 74
tnnnctatc 9
<210> 75
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 75
cnnnctatc 9
<210> 76
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 76
gnnnctatc 9
<210> 77
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 77
nannctatc 9
<210> 78
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 78
ntnnctatc 9
<210> 79
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 79
ncnnctatc 9
<210> 80
<211> 9
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 80
ngnnctatc 9
Claims (8)
1. A method for constructing an in-situ sequencing library for realizing in-situ detection of multiple RNAs is characterized by comprising the following steps of: the method comprises the following steps:
(1) designing at least one recognition probe pair according to a target sequence on the target RNA; each recognition probe pair consists of an upstream recognition probe and a downstream recognition probe, wherein the upstream recognition probe contains an upstream label sequence, the downstream recognition probe contains a downstream label sequence, and the upstream label sequence and the downstream label sequence form a complete label sequence for reading the corresponding target sequence;
(2) delivering the at least one recognition probe pair into the cell, and enabling the recognition probe pair to be recognized and hybridized with the target sequence on the target RNA, wherein the 3 'end of the upstream recognition probe and the 5' end of the downstream recognition probe are close to each other and are connected through DNA ligase capable of connecting DNA by taking RNA as a template to form at least one chain DNA molecule;
(3) hybridizing at least one chain DNA molecule obtained in the step (2) with a DNA splint sequence to enable the 5 'end and the 3' end of the at least one chain DNA molecule to be close to each other, and then connecting the at least one chain DNA molecule and the DNA splint sequence through DNA ligase to form at least one circular DNA molecule;
(4) and performing rolling circle amplification by taking the at least one annular DNA molecule as a template to obtain amplification products, namely the in-situ sequencing library, decoding different labels in the in-situ sequencing library by using an in-situ sequencing technology to obtain a complete label sequence on each amplification product, and further obtaining the types of target RNAs detected by different amplification products, so that in-situ expression information of different target RNAs is obtained, and the in-situ detection of the multiple RNAs is realized.
2. The method of construction of claim 1, wherein: the target RNA includes RNA of the cell and RNA expressed by exogenous genes in the cell.
3. The method of construction of claim 1, wherein: the DNA ligase is Splint R ligase.
4. The method of construction of claim 1, wherein: the cells are free cells to be detected or cells in tissues.
5. The method of construction of claim 1, wherein: the target RNA comprises RNA of a cell and RNA expressed by an exogenous gene in the cell, and the cell is a free cell to be detected or a cell in a tissue.
6. The method of construction of claim 1, wherein: the target RNA comprises RNA of a cell and RNA expressed by an exogenous gene in the cell, and the DNA ligase is Splint R ligase.
7. The method of construction of claim 1, wherein: the cell is a free cell to be detected or a cell in a tissue, and the DNA ligase is Splint R ligase.
8. The method of construction of claim 1, wherein: the target RNA comprises RNA of a cell and RNA expressed by an exogenous gene in the cell, the cell is a free cell to be detected or a cell in a tissue, and the DNA ligase is Splint R ligase.
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| WO2024113164A1 (en) * | 2022-11-29 | 2024-06-06 | 深圳华大智造科技股份有限公司 | In-situ sequencing method, and method for performing area division on in-situ sequencing result |
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