CN111801428A - 一种获得单细胞mRNA序列的方法 - Google Patents
一种获得单细胞mRNA序列的方法 Download PDFInfo
- Publication number
- CN111801428A CN111801428A CN201880083669.8A CN201880083669A CN111801428A CN 111801428 A CN111801428 A CN 111801428A CN 201880083669 A CN201880083669 A CN 201880083669A CN 111801428 A CN111801428 A CN 111801428A
- Authority
- CN
- China
- Prior art keywords
- cell
- molecular
- label
- tag
- carrier
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108020004999 messenger RNA Proteins 0.000 title claims abstract description 87
- 238000000034 method Methods 0.000 title claims abstract description 80
- 239000002299 complementary DNA Substances 0.000 claims abstract description 131
- 239000012634 fragment Substances 0.000 claims abstract description 79
- 108010020764 Transposases Proteins 0.000 claims abstract description 47
- 102000008579 Transposases Human genes 0.000 claims abstract description 47
- 108020004635 Complementary DNA Proteins 0.000 claims abstract description 36
- 238000010839 reverse transcription Methods 0.000 claims abstract description 33
- 238000012165 high-throughput sequencing Methods 0.000 claims abstract description 8
- 108020004414 DNA Proteins 0.000 claims description 190
- 102000053602 DNA Human genes 0.000 claims description 134
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 85
- 238000012163 sequencing technique Methods 0.000 claims description 56
- 239000007790 solid phase Substances 0.000 claims description 55
- 230000003321 amplification Effects 0.000 claims description 36
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 36
- 238000005096 rolling process Methods 0.000 claims description 23
- 230000000295 complement effect Effects 0.000 claims description 21
- 238000012408 PCR amplification Methods 0.000 claims description 14
- 239000013076 target substance Substances 0.000 claims description 6
- 230000004907 flux Effects 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 250
- 239000011324 bead Substances 0.000 description 125
- 239000000243 solution Substances 0.000 description 80
- 238000006243 chemical reaction Methods 0.000 description 52
- 239000002773 nucleotide Substances 0.000 description 31
- 125000003729 nucleotide group Chemical group 0.000 description 31
- 239000000047 product Substances 0.000 description 29
- 239000000725 suspension Substances 0.000 description 28
- 239000000872 buffer Substances 0.000 description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 26
- 239000007853 buffer solution Substances 0.000 description 23
- 239000011534 wash buffer Substances 0.000 description 23
- 239000013598 vector Substances 0.000 description 21
- 238000005516 engineering process Methods 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 13
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 239000007788 liquid Substances 0.000 description 12
- 238000000746 purification Methods 0.000 description 12
- 238000005406 washing Methods 0.000 description 12
- 238000000137 annealing Methods 0.000 description 11
- 238000010586 diagram Methods 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- 108060002716 Exonuclease Proteins 0.000 description 9
- 239000000969 carrier Substances 0.000 description 9
- 102000013165 exonuclease Human genes 0.000 description 9
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 9
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 8
- 102000012410 DNA Ligases Human genes 0.000 description 8
- 108010061982 DNA Ligases Proteins 0.000 description 8
- 229910001629 magnesium chloride Inorganic materials 0.000 description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 230000003993 interaction Effects 0.000 description 7
- 238000002372 labelling Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 230000017105 transposition Effects 0.000 description 7
- 229920001213 Polysorbate 20 Polymers 0.000 description 6
- 230000000903 blocking effect Effects 0.000 description 6
- 230000001413 cellular effect Effects 0.000 description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000007363 ring formation reaction Methods 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 238000010561 standard procedure Methods 0.000 description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 5
- 108010012306 Tn5 transposase Proteins 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 238000009396 hybridization Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical group N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- 238000006911 enzymatic reaction Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- KWIUHFFTVRNATP-UHFFFAOYSA-N glycine betaine Chemical compound C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 4
- 239000011148 porous material Substances 0.000 description 4
- 239000011535 reaction buffer Substances 0.000 description 4
- 108010007577 Exodeoxyribonuclease I Proteins 0.000 description 3
- 102100029075 Exonuclease 1 Human genes 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 108010090804 Streptavidin Proteins 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 239000012160 loading buffer Substances 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- 108050009160 DNA polymerase 1 Proteins 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 102100034343 Integrase Human genes 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- 108091028664 Ribonucleotide Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000007984 Tris EDTA buffer Substances 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 229960003237 betaine Drugs 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 238000003320 cell separation method Methods 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000004205 dimethyl polysiloxane Substances 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000001001 laser micro-dissection Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 125000001921 locked nucleotide group Chemical group 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000012264 purified product Substances 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 239000002336 ribonucleotide Substances 0.000 description 2
- 125000002652 ribonucleotide group Chemical group 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010046914 Exodeoxyribonuclease V Proteins 0.000 description 1
- -1 Polydimethylsiloxane Polymers 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 229920001486 SU-8 photoresist Polymers 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000013090 high-throughput technology Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- UEGPKNKPLBYCNK-UHFFFAOYSA-L magnesium acetate Chemical compound [Mg+2].CC([O-])=O.CC([O-])=O UEGPKNKPLBYCNK-UHFFFAOYSA-L 0.000 description 1
- 239000011654 magnesium acetate Substances 0.000 description 1
- 235000011285 magnesium acetate Nutrition 0.000 description 1
- 229940069446 magnesium acetate Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 230000008450 motivation Effects 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 238000009832 plasma treatment Methods 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000011176 pooling Methods 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- GUUBJKMBDULZTE-UHFFFAOYSA-M potassium;2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid;hydroxide Chemical group [OH-].[K+].OCCN1CCN(CCS(O)(=O)=O)CC1 GUUBJKMBDULZTE-UHFFFAOYSA-M 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 238000012174 single-cell RNA sequencing Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6816—Hybridisation assays characterised by the detection means
- C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
- C12Q2565/50—Detection characterised by immobilisation to a surface
- C12Q2565/514—Detection characterised by immobilisation to a surface characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种获得单细胞mRNA序列的方法。本发明的方法：(1)利用细胞标签载体捕获细胞的mRNA，反转录得到具有细胞标签的cDNA；来自于同一个细胞的cDNA具有相同的细胞标签，来自于不同细胞的cDNA具有不同的细胞标签；(2)利用转座酶复合体和分子标签载体，得到具有分子标签的多个cDNA片段；来自于同一cDNA的各个片段具有相同的分子标签，来自于不同cDNA的片段具有不同的分子标签；(3)高通量测序；(4)根据分子标签进行序列拼接，得到每个mRNA的序列；根据细胞标签，得到每个单细胞的所有mRNA的序列。本发明提供的方法可用于高通量获得大量单细胞中每个单细胞的所有mRNA的序列。
Description
PCT国内申请，说明书已公开。
Claims (38)
- PCT国内申请，权利要求书已公开。
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2017119342 | 2017-12-28 | ||
| CNPCT/CN2017/119342 | 2017-12-28 | ||
| PCT/CN2018/124293 WO2019129133A1 (zh) | 2017-12-28 | 2018-12-27 | 一种获得单细胞mRNA序列的方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111801428A true CN111801428A (zh) | 2020-10-20 |
| CN111801428B CN111801428B (zh) | 2023-05-23 |
Family
ID=67066601
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201880083669.8A Active CN111801428B (zh) | 2017-12-28 | 2018-12-27 | 一种获得单细胞mRNA序列的方法 |
Country Status (4)
| Country | Link |
|---|---|
| US (1) | US11718872B2 (zh) |
| EP (1) | EP3733865A4 (zh) |
| CN (1) | CN111801428B (zh) |
| WO (1) | WO2019129133A1 (zh) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113106150A (zh) * | 2021-05-12 | 2021-07-13 | 浙江大学 | 一种超高通量单细胞测序方法 |
| CN115198000A (zh) * | 2022-07-21 | 2022-10-18 | 北京寻因生物科技有限公司 | 一种单细胞全序列转录组文库构建的方法 |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114854827A (zh) * | 2022-05-18 | 2022-08-05 | 苏州极客基因科技有限公司 | 一种用于富集mRNA的磁珠复合物及其应用 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104032377A (zh) * | 2014-06-30 | 2014-09-10 | 北京诺禾致源生物信息科技有限公司 | 单细胞转录组测序文库的构建方法及其应用 |
| US20140272965A1 (en) * | 2013-03-13 | 2014-09-18 | Denovo Sciences, Inc. | System and method for capturing and analyzing cells |
| US20160304952A1 (en) * | 2015-04-14 | 2016-10-20 | Massachusetts Institue Of Technology | In situ nucleic acid sequencing of expanded biological samples |
| CN106754904A (zh) * | 2016-12-21 | 2017-05-31 | 南京诺唯赞生物科技有限公司 | 一种cDNA的特异性分子标签及其应用 |
| CN106795651A (zh) * | 2014-06-26 | 2017-05-31 | 伊鲁米那股份有限公司 | 利用单管添加方案的加标签的核酸的文库制备 |
| CN107502607A (zh) * | 2017-06-20 | 2017-12-22 | 浙江大学 | 一种大量组织、细胞样本mRNA的分子条形码标记、文库构建、测序的方法 |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9429500B2 (en) * | 2012-02-29 | 2016-08-30 | Fluidigm Corporation | Methods, systems and devices for multiple single-cell capturing and processing using microfluidics |
| US20160122753A1 (en) * | 2013-06-12 | 2016-05-05 | Tarjei Mikkelsen | High-throughput rna-seq |
| US10975371B2 (en) * | 2014-04-29 | 2021-04-13 | Illumina, Inc. | Nucleic acid sequence analysis from single cells |
| CN106498040B (zh) * | 2016-10-12 | 2018-10-23 | 浙江大学 | 一种分子标记微珠及基于该分子标记微珠的高通量单细胞测序方法 |
-
2018
- 2018-12-27 EP EP18896527.1A patent/EP3733865A4/en active Pending
- 2018-12-27 WO PCT/CN2018/124293 patent/WO2019129133A1/zh unknown
- 2018-12-27 CN CN201880083669.8A patent/CN111801428B/zh active Active
-
2020
- 2020-06-23 US US16/909,366 patent/US11718872B2/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140272965A1 (en) * | 2013-03-13 | 2014-09-18 | Denovo Sciences, Inc. | System and method for capturing and analyzing cells |
| CN106795651A (zh) * | 2014-06-26 | 2017-05-31 | 伊鲁米那股份有限公司 | 利用单管添加方案的加标签的核酸的文库制备 |
| CN104032377A (zh) * | 2014-06-30 | 2014-09-10 | 北京诺禾致源生物信息科技有限公司 | 单细胞转录组测序文库的构建方法及其应用 |
| US20160304952A1 (en) * | 2015-04-14 | 2016-10-20 | Massachusetts Institue Of Technology | In situ nucleic acid sequencing of expanded biological samples |
| CN106754904A (zh) * | 2016-12-21 | 2017-05-31 | 南京诺唯赞生物科技有限公司 | 一种cDNA的特异性分子标签及其应用 |
| CN107502607A (zh) * | 2017-06-20 | 2017-12-22 | 浙江大学 | 一种大量组织、细胞样本mRNA的分子条形码标记、文库构建、测序的方法 |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113106150A (zh) * | 2021-05-12 | 2021-07-13 | 浙江大学 | 一种超高通量单细胞测序方法 |
| CN113106150B (zh) * | 2021-05-12 | 2022-05-24 | 浙江大学 | 一种超高通量单细胞测序方法 |
| CN114958996A (zh) * | 2021-05-12 | 2022-08-30 | 浙江大学 | 一种超高通量单细胞测序试剂组合 |
| CN115011679A (zh) * | 2021-05-12 | 2022-09-06 | 浙江大学 | 一种超高通量单细胞测序方法 |
| CN114958996B (zh) * | 2021-05-12 | 2022-12-20 | 浙江大学 | 一种超高通量单细胞测序试剂组合 |
| CN115198000A (zh) * | 2022-07-21 | 2022-10-18 | 北京寻因生物科技有限公司 | 一种单细胞全序列转录组文库构建的方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| EP3733865A4 (en) | 2021-09-08 |
| US20200318181A1 (en) | 2020-10-08 |
| EP3733865A1 (en) | 2020-11-04 |
| WO2019129133A1 (zh) | 2019-07-04 |
| CN111801428B (zh) | 2023-05-23 |
| US11718872B2 (en) | 2023-08-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11591652B2 (en) | System and methods for massively parallel analysis of nucleic acids in single cells | |
| US20190203204A1 (en) | Methods of De Novo Assembly of Barcoded Genomic DNA Fragments | |
| EP4001427A1 (en) | Methods for sorting nucleic acids and multiplexed preparative in vitro cloning | |
| WO2021046232A1 (en) | Optically readable barcodes and systems and methods for characterizing molecular interactions | |
| US10876106B2 (en) | Gene analysis system | |
| CN113604545A (zh) | 一种超高通量单细胞染色质转座酶可及性测序方法 | |
| EP3615683B1 (en) | Methods for linking polynucleotides | |
| CN115198000B (zh) | 一种单细胞全序列转录组文库构建的方法 | |
| US11718872B2 (en) | Method for obtaining single-cell mRNA sequence | |
| EP3998338B1 (en) | Method for amplifying nucleic acid using solid-phase carrier | |
| CN114729349A (zh) | 条码化核酸用于检测和测序的方法 | |
| CN113026111A (zh) | 用于构建人单细胞tcr测序文库的试剂盒及其应用 | |
| JP2009284834A (ja) | 大規模並列核酸分析方法 | |
| CN116949132A (zh) | 一种构建单细胞测序文库的方法 | |
| CN115261389A (zh) | 一种功能序列、包含其的标签群以及应用 | |
| WO2018134907A1 (ja) | 同一細胞複数生体分子抽出装置および方法 | |
| US20240191299A1 (en) | Chemical sample indexing for high-throughput single-cell analysis | |
| CN112996925A (zh) | 用于crispr的靶标无关的向导rna | |
| WO2023137645A1 (en) | Adjustable droplets distribution | |
| US20220373544A1 (en) | Methods and systems for determining cell-cell interaction | |
| Smibert et al. | ECCITE-seq | |
| WO2024250155A1 (zh) | 一种构建单细胞测序文库的方法 | |
| CN119411232A (zh) | 一种基于微流控的高通量单细胞转录组测序建库方法 | |
| CN117625595A (zh) | 染色质可及性/转录组共检测试剂盒及其应用 | |
| CN120153092A (zh) | 条码化核酸用于检测和测序的方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 518083 the comprehensive building of Beishan industrial zone and 11 2 buildings in Yantian District, Shenzhen, Guangdong. Applicant after: Shenzhen Huada Zhizao Technology Co.,Ltd. Address before: 518083 the comprehensive building of Beishan industrial zone and 11 2 buildings in Yantian District, Shenzhen, Guangdong. Applicant before: MGI TECH Co.,Ltd. |
|
| CB02 | Change of applicant information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |