CN109251961A - A kind of method of unicellular sequencing detection activating T cell - Google Patents
A kind of method of unicellular sequencing detection activating T cell Download PDFInfo
- Publication number
- CN109251961A CN109251961A CN201810691767.1A CN201810691767A CN109251961A CN 109251961 A CN109251961 A CN 109251961A CN 201810691767 A CN201810691767 A CN 201810691767A CN 109251961 A CN109251961 A CN 109251961A
- Authority
- CN
- China
- Prior art keywords
- cell
- unicellular
- sequencing
- cdna
- reverse transcription
- 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.)
- Pending
Links
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
- C12Q1/6806—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
- C12Q1/6869—Methods for sequencing
Abstract
The invention discloses a kind of methods using unicellular sequencing detection activating T cell, comprising the following steps: (1) obtains the tumor-specific cytotoxicity T lymphocyte (cytotoxic lymphocyte, CTL) through heterologous gene hybrid cell induced amplification；(2) it separates unicellular and is cracked；(3) reverse transcription is carried out to the mRNA of sample and obtains cDNA；(4) PCR amplification is carried out by template of cDNA, product purification carries out Tagmentation reaction, completes the building of cDNA sequencing library；(5) high-flux sequence is carried out to the cDNA library of building；(6) sequencing result carries out bioinformatic analysis, detects T cell activation degree.
Description
Technical field
The invention belongs to fields of biomedicine, more particularly, to a kind of method of unicellular sequencing detection activating T cell.
Background technique
T cell is one kind of lymphocyte, and important role is play in immune response.T cell is broken up in thymus gland
Maturation, maturation, which moves back, to be occupy in peripheral lymphoid tissues.T cell is mainly to resist exotic invasive object and have various biology
Function.
T cell treatment is the core means of cellular immunotherapy, the side for being considered most prospect, being most hopeful tumors destroyed
Method.However, there is not strong activity, lazy weight, cannot assembling, to tumor locus without effective curative effect in T cell used at present
The problems such as monitoring means.Therefore, around these crucial matter of science and technology, we are by releasing t cell suppressor factor PD- early period
1, construct active t cell；Expanded with heterologous gene hybrid cell and T cell and assign its anticancer property, thus obtain it is more,
The stronger CTLs of activity, plays more preferably efficient anticarcinogenic effect.However, using traditional method, (flow cytomery is thin
Cell factor for secreting etc. after cellular surface anakmetomeres, cell killing assay, ELISA detection cell activation) in vitro to being lured
The activation degree of raw CTLs is detected, and there is a problem of that testing result is larger by time and spacial influence.
For same cell under different growth periods and growing environment, expression conditions are not exactly the same, lists
Cell sequencing technologies can be expanded and be sequenced to full transcript profile in individual cell level, and base in cell is studied in integral level
Because of the case where transcribing and the rule of transcriptional control.Therefore, using unicellular sequencing technologies and bioinformatic analysis technology, to T
Cell is analyzed, and the gene expression status of integral level and gene structure information in its individual cells can be preferably studied,
Understand its genetic transcription situation, transcriptional control rule and verified, being can more early stage, more accurately understanding T cell activation shape
State provides new way, new method.
Summary of the invention
It is an object of the invention to be directed to above-mentioned problem in science, it is thin to provide a kind of unicellular sequencing detection activation T
The method of born of the same parents.
Technical solution of the present invention is summarized as follows:
The method of unicellular sequencing detection activating T cell of the invention, comprising the following steps:
1) the tumor-specific CTL s through heterologous gene hybrid cell induced amplification is obtained；
2) separate unicellular and cracked: separation is unicellular, is added in lysis buffer and is cracked；
3) reverse transcription is carried out to the mRNA of sample and obtains cDNA: tried using the SuperScript of ThermoFisher company of the U.S.
Agent box construct reverse transcription system, to above-mentioned steps 2) obtain cell pyrolysis liquid carry out reverse transcription, obtain cDNA；
4) PCR amplification is carried out by template of cDNA, constructs sequencing library, is sequenced: using Illumina company of the U.S.
NexteraXT kit, to specifications to above-mentioned steps 3) obtained reverse transcription product expanded and purified, construct high pass
Measure sequencing library；Sequencing is delivered into library after the completion；
5) bioinformatic analysis is carried out to sequencing result, detects the activation degree of T cell.
Preferably, in step 1) described above, the tumor-specific CTL s of external evoked amplification is subjected to unicellular sequencing,
Unicellular activation degree is detected from mRNA level in-site.
Present invention substantive distinguishing features outstanding and significant progress are:
By the Activation using unicellular sequencing technologies early stage, accurate understanding T cell, by the tumour of external evoked amplification
Specific CTL s carries out unicellular sequencing, unicellular activation degree is detected from mRNA level in-site, compared with conventional method such as fluidic cell
The cell factor etc. secreted after instrument detection cell surface activation molecule, cell killing assay, ELISA detection cell activation, more
In early days, quickly, accurately.
Detailed description of the invention
Fig. 1 is reverse transcription and the schematic diagram for constructing sequencing library.
Fig. 2 is that secondary magnetic cell separating method sorts mouse memory CD8+The result figure of T cell.
Fig. 3 is the mouse memory CD8 obtained after purification+T cell laser co-focusing verification result figure.
Fig. 4 is the fragment size distribution figure of cDNA library before being reacted using 6%PAGE gel detection label.
Fig. 5 is using library fragment size distribution figure after the reaction of 6%PAGE gel detection label and jointing.
Fig. 6 uses library after the 2100 Bioanalyzer detection label reaction of U.S. Agilent company and jointing
Fragment size distribution figure.
Specific embodiment
Elaborate below to implementation operation of the invention, the present embodiment under the premise of the technical scheme of the present invention into
Row is implemented, and the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following realities
Apply case.
The present invention is further illustrated combined with specific embodiments below.
Referring to shown in attached drawing 1, the method for the invention using unicellular sequencing detection activating T cell is specific as follows:
1. the novel gene editing technique of application from wound efficiently knocks out the PD-1 gene of T cell.
2. the efficient heterologous gene hybrid cell of application from wound quickly induces a large amount of high activity CTLs.With the novel base from wound
Because editing technique is by the new tumor associated antigen genes, Endoglin such as hyperacute rejection gene (pig α 1,3GT) and SMP30
Gene knock-in cancer cell prepares efficient allogeneic dendritic shape cell/cancer hybrid cell；With this hybrid cell by PD-1-T cell
Induce into CTL.
3. preparing the unicellular sample of CTL.Separate unicellular and cracked: separation is unicellular, is added in lysis buffer
It is cracked.
4. the mRNA of pair sample carries out reverse transcription and obtains cDNA: using Thermo Fisher company of the U.S.
SuperScript kit constructs reverse transcription system, carries out reverse transcription to the cell pyrolysis liquid that step 3) obtains, obtains cDNA.
5. being expanded by template of cDNA, sequencing library is constructed, is sequenced, uses Illumina company of the U.S.
Nextera XT kit, is expanded and is purified to the reverse transcription product that step 4) obtains to specifications, and building is high-throughput
Sequencing library；Sequencing is delivered into library after the completion.
6. understanding the Activation of T cell by bioinformatic analysis.
Embodiment 1
The present embodiment selection mouse (Mus musculus) it is experimental material, to its Memorability CD8+T cell has carried out unicellular
The sequencing of transcript profile.
1. obtaining T cell in spleen:
1) mouse cervical dislocation is put to death, and is soaked in 5 min in 75% alcohol.
2) sterile working taking-up mouse spleen is placed in the plate for filling PBS in super-clean bench, takes two 1 ml syringes,
Syringe needle rolled over tweezers in 90 °, fixed spleen, one draw needle point and punctures spleen coating, then with needle wall by spleen cell
It is scraped from both ends；Spleen cell is blown and beaten into cell suspension repeatedly with dropper.
3) cell suspension is then moved into 50 ml centrifuge tubes, 1200 rpm/min are centrifuged 6 min；Supernatant is discarded, is played even
Cell.
4) erythrocyte cracked liquid of 1 ml is added, room temperature acts on 5 min.
5) be added 30 ml PBS dilute erythrocyte cracked liquid, by cell suspension with 200 mesh filters filter to another 50
In ml centrifuge tube, 1200 rpm/min are centrifuged 6 min.It is rinsed 2 times with PBS buffer solution.
6) count total viable cell under microscope.Cell is adjusted with the buffer after diluting in magnetic cell sorting kit
Concentration is to 1 × 108/ ml, it is spare.
2. Memorability CD8+ The extraction of T cell:
1) CD8+The purifying of T cell
It prepares buffer: taking 10 × buffer (10 × Buffer of MagCellect) of 5 ml, the sterilizing list that 45 ml are added steams
Water dilution, saves on ice.
By 1 ml mixed lymphocytes suspension (1 × 108Cell) it moves in 5 ml EP pipes, 200 μ l biotin labelings are added
CD8+ T cell mixed antibody (MagCellect Mouse CD8+ T Cell Biotinylated Antibody
Cocktail), mix gently, avoid generating bubble.4 DEG C be incubated for 15 minutes after the 250 affine magnetic beads of ul streptomysin are added
(MagCellect StrePtavidin Ferrofluid), gently blow it is even, 4 DEG C be incubated for 15 minutes after continuously add 1.55 ml
Buffer is settled to 3 ml, and gently blows even.Pay attention to there is noresidue on tube wall.EP pipe is put into magnetic frame, room temperature (18-25
DEG C) standing 6 minutes, it is seen that brown magnetic bead is mobile to tube wall.Keep EP pipe in magnetic frame, it will using pasteur pipet or pipette
Supernatant moves in 5 new ml EP pipes.EP pipe is put into magnetic frame again, supernatant is moved into new 5 after being stored at room temperature 6 minutes
In ml EP pipe, to be eliminated as much as magnetic bead.The cell finally obtained is mouse spleen CD8+ T cell, spare.
2) Memorability CD8+T cell purifying
By CD8+Supernatant is abandoned after T cell centrifugation, is resuspended with the buffer of 100 ul, the CD62L- biotin that 0.5 ul is added is anti-
Body, 4 DEG C be incubated for 15 minutes after 12.5 ul Streptavidin MagneSpheres are added, in 4 DEG C be incubated for 15 minutes.EP pipe is put into magnetic force
Frame, (18-25 DEG C) of room temperature stands 6 minutes, it is seen that brown magnetic bead is mobile to tube wall.It keeps EP pipe in magnetic frame, uses Pasteur
Supernatant is moved to 2 new ml EP and managed by suction pipe or pipette.The cell finally obtained is the mouse spleen Memorability CD8 of high-purity+T cell can be used for subsequent experimental (referring to shown in attached drawing 2 and Fig. 3).
3. separating unicellular and being cracked:
1) with RNaseZap and the solution cleaning work platform without DNA and liquid transfer gun head；
2) following reagent is mixed as lysis buffer:
A) 19 μ l of 0.2%Triton-X100 solution；
B) the 1 μ l of RNase inhibitor of 40U/ μ l.
3) 1.19 μ l lysis buffers are added into the thin-walled PCR pipe of 0.2ml；
4) separated from step (2) resulting cell with minimum volume unicellular, volume is generally 0.3 μ l, is added slow containing cracking
In the PCR pipe of fliud flushing.
4. the mRNA of pair sample carries out reverse transcription and obtains cDNA:
1) following reagent is mixed as primer-dNTP premixed liquid:
A) dNTP(10mM) 10 μ l；
B) Oligo-dT3010 μ l of VN primer (10 μM).
2) following reagent is mixed as reverse transcription premixed liquid:
A) the 2 μ l of SuperScript III first-strand buffer of U.S. ThermoFisher company；
B) the 0.5 μ l of SuperScript III reverse transcriptase solution of U.S. ThermoFisher company；
C) 0.25 μ l of RNAse inhibitor (40U/ μ l)；
D) 0.5 μ l of DTT solution (100mM)；
E) 2 μ l of alkali solution of beet (5M)；
F) 0.06 μ l of magnesium chloride solution (1M)；
G) 0.2 μ l of TSO solution (100 μM)；
H) 0.5 μ l of ERCC exogenous RNA standard sample solution.
3) 2 μ l primer-dNTP premixed liquids are added in the resulting PCR pipe containing cell and lysis buffer of step (3)；
4) the concussion PCR pipe that is quickly vortexed is sufficiently mixed solution, is immediately placed on ice after being centrifuged 10s at room temperature with 700g；
5) it with 72 DEG C of samples of incubation 3min, puts back to immediately on ice；
6) 10s is centrifuged with 700g at room temperature, collects the liquid of PCR pipe bottom, places it on ice；
7) reverse transcription premixed liquid is added in unicellular pyrolysis product, using pipettor, gently pressure-vaccum mixes sample several times, mixes
It avoids generating bubble in the process；
8) 10s is centrifuged with 700g at room temperature, collects the liquid of PCR pipe bottom；
9) PCR pipe with sample is placed in PCR instrument, setting program carries out the reverse transcription of cell mRNA, obtains the first chain of cDNA
The thermal cycle conditions of reaction product, reverse transcription process are shown in Table 1 below.
Table 1: the thermal cycle conditions of reverse transcription process
5. being expanded by template of cDNA, sequencing library is constructed, is sequenced:
1) following solution is mixed as PCR mixed liquor:
A) 10 μ l of the first chain reaction product obtained in step (4)；
B) the 12.5 μ l of KAPA HiFi HotStart ReadyMix solution of U.S. Roche Molecular Systems company；
C) 0.25 μ l of ISPCR primer solution (10 μM)；
D) 2.25 μ l of nuclease-free water.
2) PCR mixed liquor in 15 μ l step 1) is taken to be added in PCR pipe, the concussion that is vortexed mixes sample, at room temperature with
700g is centrifuged 10s, collects the liquid of PCR pipe bottom；
3) PCR pipe with sample is placed in PCR instrument, setting program carries out the amplification of cell cDNA, the thermal cycle conditions of program
It is shown in Table 2 below；
The thermal cycle conditions of table 2:cDNA amplification program
4) the AMPure XP magnetic bead of Beckman Coulter company of the U.S. is placed into 15min at room temperature, the concussion that is vortexed is mixed
It is even；
5) 25 μ l magnetic beads are added in isometric obtained cDNA amplified production of step 3), are mixed with pipettor pressure-vaccum 10 times,
It transfers the solution into 96 orifice plates, is incubated at room temperature 8min；
6) 96 orifice plates are placed on magnetic frame 5min, is clarified to solution, carefully removes supernatant；80% ethyl alcohol of 200 μ l is added
Magnetic bead is cleaned, ethyl alcohol will be carefully removed after being incubated for 30s, be repeated 2 times；
7) after removing completely ethyl alcohol, 5min is placed at room temperature, and after one of crack occur in magnetic bead surfaces, the EB that 15 μ l are added is molten
Liquid is mixed with pipettor pressure-vaccum 10 times；
8) magnetic frame is removed, 2min is placed at room temperature for；96 orifice plates are put back on magnetic frame again, are placed 2 minutes, after solution clarification,
The supernatant for drawing 13 μ l moves on in a new 0.2ml thin-walled PCR pipe；
9) using 6% PAGE gel detection cDNA amplified production fragment size distribution, good library should clip size should
Between 500 ~ 3000bp, it is not less than the short-movie section of 500bp, and peak should appear between 1500 ~ 2000bp (referring to attached
Shown in Fig. 4)；
10) following solution is mixed as label premixed liquid:
A) the 4 μ l of marker DNA buffer of U.S. Illumina company；
B) the 1.67 μ l of label enzyme mixation of U.S. Illumina company；
C) the 1.33 μ l of resuspension buffer of U.S. Illumina company.
11) cDNA obtained in 1 μ l step 8) is taken, the label premixed liquid in 7 μ l step 10) is added, is incubated at 55 DEG C
CDNA is marked in 10min；
12) the NT buffer for adding the Illumina company of the U.S. of 2 μ l is incubated at room temperature 5min, completes label reaction；
13) following solution is mixed as connector ligation amplification premixed liquid:
A) the 15 μ l of KAPA HiFi HotStart ReadyMix solution of U.S. Roche Molecular Systems company；
B) 1 primer solution of Index, the 2.5 μ l of U.S. Illumina company；
C) 2 primer solution of Index, the 2.5 μ l of U.S. Illumina company.
14) the connector connection premixed liquid in 20 μ l step 13) is added into 10 μ l marked products obtained in step 12),
It is placed in PCR instrument, setting program carries out the connection of connector, and the thermal cycle conditions of program are shown in Table 3 below.
Table 3: the thermal cycle conditions of connector linker
15) the AMPure XP magnetic bead of Beckman Coulter company of the U.S. is placed into 15min at room temperature, the concussion that is vortexed is mixed
It is even；
16) 24 μ l magnetic beads are added in the 30 μ l products that step 13) obtains according to the volume ratio of 0.8:1, with pipettor pressure-vaccum 10
Secondary mixing transfers the solution into 96 orifice plates, is incubated at room temperature 8min；
17) step 6) is repeated to step 8), purifies amplified production；
18) using 6% PAGE gel and Agilent company of the U.S. 2100 Bioanalyzer detecting steps 17) in surveyed
The fragment size distribution (referring to shown in attached drawing 5 and Fig. 6) in preface library, and it is glimmering with the Qubit3.0 of ThermoFisher company of the U.S.
Photometry measures the concentration of each library DNA；
19) it according to the data volume of the concentration and required sequencing that measure each library in step 18, calculates each in final library mixed liquor
The molar ratio in library, in proportion mixes library, is measured using Qubit3.0 fluorimeter, and adjustment final concentration reaches 5nM；
20) sequencing company is sent to carry out high-flux sequence.
Selection mouse (Musmusculus) Memorability CD8+T cell is experimental material merely to citing, it was demonstrated that this side
Method can be successfully applied to the unicellular transcript profile sequencing of mammal activating T cell, but the present invention can be applied equally to people
The unicellular transcript profile of the activating T cell of class and other mammals is sequenced.
The invention is not limited to specific embodiment above-mentioned, the present invention expand to disclosed in any this specification it is new
Feature or new combination, and disclose any new method or process the step of or new combination.
Claims (2)
1. a kind of method of unicellular sequencing detection activating T cell, it is characterised in that: the following steps are included:
1) the tumor-specific CTL s through heterologous gene hybrid cell induced amplification is obtained；
2) separate unicellular and cracked: separation is unicellular, is added in lysis buffer and is cracked, obtains cell cracking
Liquid；
3) reverse transcription is carried out to the mRNA of sample and obtains cDNA: tried using the SuperScript of ThermoFisher company of the U.S.
Agent box construct reverse transcription system, to above-mentioned steps 2) obtain cell pyrolysis liquid carry out reverse transcription, obtain cDNA；
4) PCR amplification is carried out by template of cDNA, constructs sequencing library, is sequenced: using Illumina company of the U.S.
NexteraXT kit, to above-mentioned steps 3) obtained reverse transcription product expanded and purified, building high-flux sequence text
Library；Sequencing is delivered into library after the completion；
5) bioinformatic analysis is carried out to sequencing result, detects the activation degree of T cell.
2. the method for unicellular sequencing detection activating T cell according to claim 1, it is characterised in that: in the step 1),
The tumor-specific CTL s of external evoked amplification is subjected to unicellular sequencing, T cell activation degree is detected from mRNA level in-site.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810691767.1A CN109251961A (en) | 2018-06-28 | 2018-06-28 | A kind of method of unicellular sequencing detection activating T cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810691767.1A CN109251961A (en) | 2018-06-28 | 2018-06-28 | A kind of method of unicellular sequencing detection activating T cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109251961A true CN109251961A (en) | 2019-01-22 |
Family
ID=65050761
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810691767.1A Pending CN109251961A (en) | 2018-06-28 | 2018-06-28 | A kind of method of unicellular sequencing detection activating T cell |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109251961A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022121742A1 (en) * | 2020-12-07 | 2022-06-16 | 中国科学院脑科学与智能技术卓越创新中心 | Single cell transcriptome sequencing method |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2875695A1 (en) * | 2012-06-15 | 2013-12-19 | The Board Of Regents Of The University Of Texas System | High throughput sequencing of multiple transcripts of a single cell |
| CN104560883A (en) * | 2015-01-04 | 2015-04-29 | 广西医科大学 | Cancer cell/DC fusion tumor vaccine for expressing Endoglin and preparation method for cancer cell/DC fusion tumor vaccine |
| CN106957905A (en) * | 2016-12-23 | 2017-07-18 | 孙涛 | A kind of molecular detecting method and Primer composition and kit for being used to assess immunotherapy of tumors effect |
| CN107002076A (en) * | 2014-09-15 | 2017-08-01 | 阿布维特罗有限责任公司 | High flux nucleic acid libraries are sequenced |
| CN108103055A (en) * | 2018-01-09 | 2018-06-01 | 上海亿康医学检验所有限公司 | A kind of method of unicellular RNA reverse transcriptions and library construction |
-
2018
- 2018-06-28 CN CN201810691767.1A patent/CN109251961A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2875695A1 (en) * | 2012-06-15 | 2013-12-19 | The Board Of Regents Of The University Of Texas System | High throughput sequencing of multiple transcripts of a single cell |
| CN107002076A (en) * | 2014-09-15 | 2017-08-01 | 阿布维特罗有限责任公司 | High flux nucleic acid libraries are sequenced |
| CN104560883A (en) * | 2015-01-04 | 2015-04-29 | 广西医科大学 | Cancer cell/DC fusion tumor vaccine for expressing Endoglin and preparation method for cancer cell/DC fusion tumor vaccine |
| CN106957905A (en) * | 2016-12-23 | 2017-07-18 | 孙涛 | A kind of molecular detecting method and Primer composition and kit for being used to assess immunotherapy of tumors effect |
| CN108103055A (en) * | 2018-01-09 | 2018-06-01 | 上海亿康医学检验所有限公司 | A kind of method of unicellular RNA reverse transcriptions and library construction |
Non-Patent Citations (2)
| Title |
|---|
| WEI ZHANG等: "Genomic expression analysis by single-cell mRNA differential display of quiescent CD8 T cells from tumour-infiltrating lymphocytes obtained from in vivo liver tumours", 《IMMUNOLOGY》 * |
| 周超平等: "单细胞转录组研究进展", 《生物化学与生物物理进展》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022121742A1 (en) * | 2020-12-07 | 2022-06-16 | 中国科学院脑科学与智能技术卓越创新中心 | Single cell transcriptome sequencing method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Han et al. | Mapping the mouse cell atlas by microwell-seq | |
| US11053543B2 (en) | System and methods for massively parallel analysis of nucleic acids in single cells | |
| US20210087549A1 (en) | Single-cell freeze-thaw lysis | |
| EP2861760B1 (en) | High throughput sequencing of multiple transcripts of a single cell | |
| EP3420102B1 (en) | Methods for identifying and modulating immune phenotypes | |
| CA2906076A1 (en) | Single cell bar-coding for antibody discovery | |
| WO2019086007A1 (en) | Sgrna for targeting and guiding cas9 protein to efficiently cleave tcr and b2m gene loci | |
| JP2009284834A (en) | Large-scale parallel nucleic acid analysis method | |
| CN109251961A (en) | A kind of method of unicellular sequencing detection activating T cell | |
| Tanizawa et al. | Unravelling global genome organization by 3C-seq | |
| CN109576356A (en) | A kind of method of unicellular sequencing screening lymphatic endothelial cells mark molecule | |
| CN109576355A (en) | A kind of method of unicellular sequencing screening tumor vascular endothelial cell mark molecule | |
| US20220333200A1 (en) | In vitro fertilisation (ivf) embryo viability and quality assay | |
| Daum et al. | The microRNA processing subunit DGCR8 is required for a T cell-dependent germinal center response | |
| CN104774872B (en) | Change the method and its application of cell fate | |
| Yates et al. | SLALOM: A Simple and Rapid Method for Enzymatic Synthesis of CRISPR-Cas9 sgRNA Libraries | |
| US20220373544A1 (en) | Methods and systems for determining cell-cell interaction | |
| Rees | Heterogeneity and Impaired IGF1R Signalling in Aged B cell Development | |
| Liu et al. | The utilization of single-cell sequencing technology in investigating the immune microenvironment of ccRCC | |
| WO2023239733A1 (en) | Combinatorial indexing for single-cell nucleic acid sequencing |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20190122 |
|
| WD01 | Invention patent application deemed withdrawn after publication |