CN109762728A - A kind of space transcript profile detection chip and method - Google Patents
A kind of space transcript profile detection chip and method Download PDFInfo
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- CN109762728A CN109762728A CN201910013293.XA CN201910013293A CN109762728A CN 109762728 A CN109762728 A CN 109762728A CN 201910013293 A CN201910013293 A CN 201910013293A CN 109762728 A CN109762728 A CN 109762728A
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Abstract
The present invention provides a kind of space transcript profile sequence testing chip and method, the chip and method are for measuring one of different spatial or a variety of unicellular sequences in tissue；Including microwell plate and coding sequencing microballoon, microwell plate is equipped with coding sequencing microballoon；The method includes using a variety of random codeds and microballoon to be coupled, each of them random coded includes molecular labeling and position mark bar code；Molecular labeling can detecte the sequence information of tissue monocytes, and position mark is used to the unicellular spatial positional information of record organization；It can analyze detection tissue monocytes specific transcriptional group information based on the detection chip and method, and can detect thousands of single celled spatial positions in above-mentioned tissue simultaneously.
Description
Technical field
The present invention relates to field of biotechnology, more particularly to a kind of space transcript profile sequence testing chip and method.
Background technique
Traditional transcript profile research, in most cases carries out both for a large amount of cells of mixing, can not observe
To fine distinction between individual cells, it is poor to mask specificity and cell in the behavior and biological phenomena of independent individual sample
It is anisotropic.And it is directed to the research of individual cells, be the limiting condition that cell life analytical technology is pursued, developed to traditional technology
Ultimate challenge.Many cells group may include foreign cell environment such as tissue and tumour.These complicated cellular environments usually may be used
To show a variety of phenotypes, these phenotypes can indicate Multi-genotype.It is refined from many cells complexity to unicellular variable
Property is the importance for understanding many cells heterogeneity.
Currently, the RNA sequencing that tissue transcript profile usually passes through homogeneous biopsy is studied, histocyte space letter is resulted in
The missing of breath.Or there are certain methods to carry out cell position position monitor, but they have in analyzable transcript quantity
Limit, and dependent on a large amount of available data collection, expensive instrument or heavy workload.But determine the identity of sample target
Clinical application, diagnostics and biomedical research are important with position.Accordingly, there exist to can make target molecule in sample
The demand of the associated methods and techniques in position in product.
Summary of the invention
In order to meet the demand, there is provided herein a kind of space transcript profile sequence testing chip and method, the chip and method
For measuring single celled sequence information and spatial positional information in tissue.
Herein the technical solution adopted is that:
A kind of detection chip of space transcript profile, including microwell plate and coding sequencing microballoon, microwell plate are equipped with coding sequencing
Microballoon；The space bar code information of microballoon is sequenced by label random coded come the single celled space bit of record organization in the method
Confidence breath.
Preferably, the coding sequencing microballoon is that microballoon ontology and oligonucleotide chain carry out coupling reaction generation, few nucleosides
Sour chain carries out coupling reaction as free token and molecular labeling.
Preferably, the oligonucleotide chain is that microballoon ontology and poly U tail carry out coupling reaction generation, as amplimer
The amplification of bond area and sequencing sequence provide the space bar code of cell spaces location information, carry out the original position of in situ hybridization
Sequencing sequence prevents deviation caused by expanding while recording the UMI of expression quantity, captures reverse transcription widow's core of mRNA in histocyte
Thuja acid primer poly T tail.
Preferably, the oligonucleotide chain includes poly U tail, amplification and sequencing primer, position bar code, sequencing is drawn in situ
Object, UMI and poly T tail.
Preferably, the preparation method of the microwell plate, the specific steps are as follows:
(1) micropore is etched on silicon wafer as initial mould, micro-pore diameter is at 10 ~ 200 μm;
(2) it in initial mould upper dimethyl silicone polymer, is removed after molding, that is, can be made into the reversed mold with microtrabeculae；
(3) it in the agarose of reversed mold upper heating and melting, is removed after cooling and shaping, that is, can be made into microwell plate.
Preferably, the microwell plate is equipped with micropore, and the coding sequencing microballoon is put into micropore；The micropore is circle
Shape, hexagon, rectangular or other passes；
Preferably, the arrangement mode of the micropore is Square array, Hexagonal array, other regular arrangements or random row
Column.
Preferably, the spacing of the micropore is 1-100 μm.
Preferably, the number of the micropore is 1 or more.
Preferably, the diameter of the microballoon ontology is 10-35 μm.
Preferably, the microballoon ontology is silica gel microball, glass microsphere, magnetic microsphere, cross-link dextran Ago-Gel
Any one in microballoon, silicon dioxide microsphere, cellulose microsphere, polystyrene microsphere or polypropylene microballoon or above combination.
Preferably, a kind of detection method of the detection chip of space transcript profile, the specific steps are as follows:
(1) preparation of coding sequencing microballoon: coding sequencing microballoon is added in microwell plate, and microwell plate is added in coding sequencing microballoon
In fall porosity greater than 99 %；Coding sequencing microballoon in microwell plate is sequenced, and records coding sequencing microballoon in micropore
It numbers, or the coding sequencing microballoon of known coded sequence is added in micropore according to sequence, and record microballoon in microwell plate in plate
In location information；Wherein microwell plate micro-pore diameter size is just to accommodate a coding microballoon is sequenced；
(2) histotomy, tissue come from Different Organs；
(3) tissue is fixed；
(4) infiltration is handled；
(5) cDNA is synthesized: tissue being impressed on equipped with the micropore board chip for placing coded sequence microballoon, is captured in histocyte
MRNA carries out reverse transcription, synthesizes cDNA；Wherein the micro-pore diameter size of microwell plate is that just receiving one is unicellular；It is unicellular
The porosity that falls that micropore is added is controlled in 40 % or more；
(6) tissue removes；
(7) microballoon discharges；
(8) sequencing library construction, sequencing, data processing and inversion: unicellular, the building sequencing with coding sequencing microballoon is collected
Library carries out histocyte sequence and spatial positional information detection.
Preferably, each space bar code battle array sequence in the coding sequencing microballoon is different.
Due to using the above technology, the present invention compared with the prior art, is had the advantage that as follows:
MRNA in analysis histotomy is the foundation stone of biomedical research and diagnosis.The present invention allows to cut in single tissue
On piece carries out visualization and quantitative analysis to the transcription with spatial resolution.By in the battle array with unique location bar code
Column inversion records position tissue slice on primer, while showing the RNA sequencing data of high quality and remains from mouse
The spatial positional information of brain.The present invention constructs a microwell array chip, realizes in situ detection histocyte sequence and position
Confidence ceases simple, not damaged, high-resolution a kind of space transcript profile detection, it can be achieved that biological tissue.
Detailed description of the invention
Fig. 1 is the schematic diagram of coding sequencing microballoon；
Fig. 2 is the schematic diagram that microwell plate is added in coding sequencing microballoon at random；
Fig. 3 is the schematic diagram of high pass quantity space transcript profile detection；
In figure: 1, microballoon ontology, 2, coding sequencing microballoon, 3, microwell plate, 4, micropore spacing, 5, tissue.
Specific embodiment
Next combining some specific implementation cases, the present invention is further elaborated, but claim of the invention is not only
It is limited only to following examples.
Embodiment 1
1, the preparation of microwell plate
3 size of microwell plate is designed according to experimental size, orifice plate size is the mm of 3 mm × 3, and micropore conduct is etched on silicon wafer
Initial mould, 20 μm of micropore depth and 15 μm of micro-pore diameter size, pitch of holes 4 are 10 μm.
Silicon wafer upper dimethyl silicone polymer (PDMS), takes down PDMS after molding, that is, can be made into the reversed of microtrabeculae
Mold, the agarose prepared with no enzyme water for being 5 % with concentration, heat are cast on reversed mold (PDMS microtrabeculae plate) after melting and condense
Molding, agarose plate peel after be exactly with certain thickness microwell plate 3.It is added on 3 surface of microwell plate to cell when preservation
Harmless DPBS-EDTA mixed liquor, capping are stored in 4 DEG C of refrigerators, can be prepared by microwell plate 3, that is, are made i.e. of can guarantee micropore
3 good working order of plate.
2, the preparation of coding sequencing microballoon
Space bar code array: 1000 oligonucleotide arrays are coupled on microballoon ontology 1, wherein each array includes one
A 18- aggressiveness unique bar code, a 9- gather semi-random molecular recognition code and a poly- 20 TVN capture oligo；And it will
Coding sequencing microballoon 2 is fixed at random in each micropore in microwell plate 3.
Space bar code sequence: UUUUUGACTCCGTAATACGAC TCACTATAGGGACACGA CGCTCTTCCGATCT
[18mer_SPATIALBARCODE]WSNNWSNNVTTTTTTTTTTTTTTTTTTTTVN
Wherein, V is any nucleotide (A/C/G) other than T, and N is any nucleotide (A/T/C/G).
3, tissue separation and preparation
(1) histotomy: adult mice brain tissue and quick-frozen is taken.Group is embedded before being woven in slice with cold OCT, is then protected in low temperature
Frozen section is carried out in holder, with a thickness of 10 μm.
(2) tissue is fixed: biopsy tissues are mounted in the microwell plate 3 where the microballoon ontology 1 of space bar code array modification
On, guarantee that each micropore organized 5 falls within top.
4, infiltration processing is synthesized with cDNA
(1) the exonuclease I buffer that 70 μ l include 0.19 μ g/ μ l BSA, and 37 DEG C of cultivations are uniformly added into micropore
30 min；
(2) each micropore in microwell plate 3 is cleaned with the SSC of 100 μ l 0.1x and secondary ultrapure water；
(3) the 0.1% pepsin (Sigma-that 70 μ l are dissolved in the HCL of 0.1 M is uniformly added into each micropore
), and 37 DEG C of 10 min of cultivation Aldrich；
(4) according to step (2) repeated washing microwell plate 3；
(5) each micropore is added 70 μ l reverse transcription mixed liquors and cultivates 6h in the microwell plate 3 after cleaning in (4).Wherein, instead
Transcribing mixed liquor includes: 1x the first chain buffer, the 5 mM μ g/ μ l of DTT, 500 μM of dNTP, 0.19 BSA, 50 ng/ μ
L Actinomycin D, 1% DMSO, 20 U/ μ l Superscript III and 2 U/ μ l RNaseOUT.
5, tissue removes
The Proteinase K and PKD buffer solution (pH 7.5) that 70 μ l volume ratios are 1:7 are added in each micropore, 56 DEG C are cultivated 1
h.Then the microwell plate 3 after tissue 5 removes is rinsed with the 2x SSC containing 0.1% SDS at 50 DEG C, then at room temperature successively
It is rinsed with 2x SSC and 1x SSC.
6, microballoon release, sequencing library building and sequencing
(1) 70 μ l release mixture is added in each micropore, 1h is cultivated under the conditions of 37 DEG C, and collect from each micropore
To the 65 μ l mixtures;Wherein, mixture includes 1.1x the second chain buffer, 8.75 μ l dNTP, 0.20 μ g/ μ l BSA
With 0.1U/ μ l USER Enzyme；
(2) 5 μ l second segment mixtures are added, and cultivate 2h under the conditions of 16 DEG C;5 μ l T4 archaeal dna polymerases then are added simultaneously
20 min are cultivated under the conditions of 16 DEG C;Wherein, second segment mixture includes 2.7x the first chain buffer, and 3.7U/ μ l DNA is poly-
Synthase I and 0.18 U/ μ l ribonuclease H；
(3) 25 μ l deionized waters are added, and are purified with Agencourt RNAClean XP pearl;
(4) sequence library based on amplification is established using in-vitro transcription technology, the sample of collection is mixed with 3 microlitres of in-vitro transcription
Close object mixing, wherein the mixture includes: 1x T7 reaction buffer, 7.5 mM NTP, 1x T7 enzyme mixations and 1 U/ μ l
SUPE Rase IN, 37 DEG C of cultivation 12h;
(5) sample to be collected to be purified using Agencourt RNAClean XP pearl, application method illustrates according to merchant product,
Then rinsed with deionized water;
(6) it collects sample to be placed on ice in 70 DEG C of heating 2min, it is slow that the reaction of 4.5 μ l 1x T4 RNA ligases is then added
Fliud flushing, 20 U/ μ l 1x T4 RNA ligase, 2,4 U/ μ l RNase inhibitor and 0.5 μM of connection adapter, and trained at 25 DEG C
1h is educated, is purified with Agencourt RNAClean XP pearl, then IDT and 1 μ l 0.83mM with 0.5 μM of 1 μ l
DNTPs mixing;
(8) sequencing library is finally diluted to 4 nM, and carries out pairing sequencing on Illumina NextSeq platform.
Embodiment 2
Adult mice is taken into its cerebral tissue after euthanasia, and histotomy preparation is carried out according to method in embodiment.
Remaining step is the same as embodiment 1.
Pairing sequencing is carried out on Illumina NextSeq platform after gene sequencing library construction, is sieved by splitting
Choosing compares, and obtains gene expression profile.
Embodiment 3
Adult mice is taken into its liver organization after euthanasia, and histotomy preparation is carried out according to method in embodiment.
Remaining step is the same as embodiment 1.
Pairing sequencing is carried out on Illumina NextSeq platform after gene sequencing library construction, is sieved by splitting
Choosing compares, and obtains gene expression profile.
Claims (10)
1. a kind of detection chip of space transcript profile, it is characterised in that: including microwell plate and coding sequencing microballoon, microwell plate
It is equipped with coding sequencing microballoon.
2. a kind of detection chip of space transcript profile as described in claim 1, it is characterised in that: the coding is sequenced microballoon and is
Microballoon ontology and oligonucleotide chain carry out coupling reaction generation.
3. a kind of detection chip of space transcript profile as claimed in claim 2, it is characterised in that: the oligonucleotide chain is micro-
Ball ontology and poly U tail carry out coupling reaction generation.
4. a kind of detection chip of space transcript profile as described in claim 1, which is characterized in that the preparation side of the microwell plate
Method, the specific steps are as follows:
(1) micropore is etched on silicon wafer as initial mould, micro-pore diameter is at 10 ~ 200 μm;
(2) it in initial mould upper dimethyl silicone polymer, is removed after molding, that is, can be made into the reversed mold with microtrabeculae；
(3) it in the agarose of reversed mold upper heating and melting, is removed after cooling and shaping, that is, can be made into microwell plate.
5. a kind of detection chip of space transcript profile as described in claim 1, it is characterised in that: the microwell plate is equipped with micro-
Hole, coding sequencing microballoon are put into micropore；The micropore is round, hexagon or rectangular, the arrangement mode of the micropore
For regular arrangement or random arrangement.
6. a kind of detection chip of space transcript profile as claimed in claim 5, it is characterised in that: the spacing of the micropore is 1-
100μm。
7. a kind of detection chip of space transcript profile as claimed in claim 5, it is characterised in that: the number of the micropore is 1
It is a or more.
8. a kind of detection chip of space transcript profile as claimed in claim 3, it is characterised in that: the diameter of the microballoon ontology
It is 10-35 μm.
9. a kind of detection chip of space transcript profile as claimed in claim 3, it is characterised in that: the microballoon ontology is silica gel
Microballoon, glass microsphere, magnetic microsphere, cross-link dextran agarose gel microsphere, silicon dioxide microsphere, cellulose microsphere, polyphenyl
Any one in ethylene microballoon or polypropylene microballoon or above combination.
10. a kind of detection method of the detection chip for space a kind of as described in claim 1-9 transcript profile, feature exist
In the specific steps are as follows:
(1) preparation of coding sequencing microballoon: coding sequencing microballoon is added in microwell plate, micro- to the coding sequencing in microwell plate
Ball is sequenced, and is recorded coding sequencing microballoon and numbered in microwell plate, or the coding sequencing microballoon of known coded sequence is pressed
It is added in micropore according to sequence, and records location information of the microballoon in microwell plate；
(2) histotomy；
(3) tissue is fixed；
(4) infiltration is handled；
(5) cDNA is synthesized: tissue being impressed on equipped with the micropore board chip for placing coded sequence microballoon, is captured in histocyte
MRNA carries out reverse transcription, synthesizes cDNA；
(6) tissue removes；
(7) microballoon discharges；
(8) sequencing library construction, sequencing, data processing and inversion: unicellular, the building sequencing with coding sequencing microballoon is collected
Library carries out histocyte sequence and spatial positional information detection.
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| CN110577983A (en) * | 2019-09-29 | 2019-12-17 | 中国科学院苏州生物医学工程技术研究所 | High-throughput single-cell transcriptome and gene mutation integration analysis method |
| CN110724733A (en) * | 2019-10-30 | 2020-01-24 | 东南大学 | Sequencing chip for high-resolution space transcriptome |
| CN110804654A (en) * | 2019-10-30 | 2020-02-18 | 东南大学 | Space transcriptome sequencing method |
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| WO2021056653A1 (en) * | 2019-09-29 | 2021-04-01 | 中国科学院苏州生物医学工程技术研究所 | Encoded chip, method and device for high-throughput integrative analysis of single-cell transcriptome and gene mutation |
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| CN113117614A (en) * | 2021-03-16 | 2021-07-16 | 苏州为度生物技术有限公司 | Magnetic microsphere with uniform particle size and preparation method thereof |
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| CN114958996A (en) * | 2021-05-12 | 2022-08-30 | 浙江大学 | Ultrahigh-flux single-cell sequencing reagent combination |
| WO2023011628A1 (en) * | 2021-08-06 | 2023-02-09 | 吉林大学 | High-resolution spatial omics detection method for tissue sample |
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| WO2021056653A1 (en) * | 2019-09-29 | 2021-04-01 | 中国科学院苏州生物医学工程技术研究所 | Encoded chip, method and device for high-throughput integrative analysis of single-cell transcriptome and gene mutation |
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| CN110724733A (en) * | 2019-10-30 | 2020-01-24 | 东南大学 | Sequencing chip for high-resolution space transcriptome |
| CN110804654A (en) * | 2019-10-30 | 2020-02-18 | 东南大学 | Space transcriptome sequencing method |
| CN110724733B (en) * | 2019-10-30 | 2022-11-11 | 东南大学 | Sequencing chip for high-resolution space transcriptome |
| CN112522371A (en) * | 2020-12-21 | 2021-03-19 | 广州基迪奥生物科技有限公司 | Analysis method of spatial transcriptome sequencing data |
| CN112858670B (en) * | 2021-01-26 | 2021-12-17 | 深圳市科瑞达生物技术有限公司 | Multiple digital ELISA detection method and microfluidic chip |
| CN112858670A (en) * | 2021-01-26 | 2021-05-28 | 深圳市科瑞达生物技术有限公司 | Multiple digital ELISA detection method and microfluidic chip |
| CN113117614A (en) * | 2021-03-16 | 2021-07-16 | 苏州为度生物技术有限公司 | Magnetic microsphere with uniform particle size and preparation method thereof |
| CN114958996A (en) * | 2021-05-12 | 2022-08-30 | 浙江大学 | Ultrahigh-flux single-cell sequencing reagent combination |
| CN114958996B (en) * | 2021-05-12 | 2022-12-20 | 浙江大学 | Ultrahigh-throughput unicellular sequencing reagent combination |
| CN113270142A (en) * | 2021-05-19 | 2021-08-17 | 东南大学 | Space transcriptome sequencing decoding method based on transient coding |
| CN113270142B (en) * | 2021-05-19 | 2024-09-24 | 东南大学 | Space transcriptome sequencing decoding method based on transient coding |
| WO2023011628A1 (en) * | 2021-08-06 | 2023-02-09 | 吉林大学 | High-resolution spatial omics detection method for tissue sample |
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