WO2023284076A1 - Procédé de séquençage du ranslatome unicellulaire - Google Patents

Procédé de séquençage du ranslatome unicellulaire Download PDF

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WO2023284076A1
WO2023284076A1 PCT/CN2021/115194 CN2021115194W WO2023284076A1 WO 2023284076 A1 WO2023284076 A1 WO 2023284076A1 CN 2021115194 W CN2021115194 W CN 2021115194W WO 2023284076 A1 WO2023284076 A1 WO 2023284076A1
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rna
complex
minutes
cell
binding molecule
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杨运桂
鞠林芳
杨莹
韩潇
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中国科学院北京基因组研究所(国家生物信息中心)
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

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  • the invention relates to a method for obtaining RNA combined with ribosomes in single cells. Further, it relates to a method for single-cell translation genome sequencing, and a method for simultaneously performing single-cell transcriptome sequencing and translation genome sequencing.
  • Ribosome profiling technology is an important means to study translational regulation at the genome level. Based on the traditional Ribo-seq method, many improved techniques have also been widely used in the study of translation regulation. These methods have their own advantages and disadvantages, and provide technical support in identifying translation initiation sites, analyzing translation cycle mechanisms, drawing active translation maps, and studying translation heterogeneity and bias.
  • Ribo-seq technologies have also been developed, but these technologies still have some disadvantages.
  • a large initial amount of cells is required (for example, 200,000 initial cells), and it is impossible to achieve trace cells and single cells. Or it cannot accurately reflect the state of translation in the cell, and the method of obtaining ribosome complexes in the cell and tissue lysate disrupts the real physiological state of the cell body, which is an in vitro reaction, and the result does not reflect the real translation of mRNA in the cell state.
  • the inventors of the present application captured RNA bound to ribosomes in single cells through a large number of experiments and repeated explorations. Based on this, single-cell translational genome sequencing was performed based on the captured RNA. Further, single-cell transcriptome sequencing and translation genome sequencing were performed simultaneously, and thus the present invention was completed.
  • the application provides a method for obtaining RNA bound to ribosomes in a single cell, the method comprising:
  • step (b) providing a binding molecule, contacting the binding molecule with the single cell obtained in step (a), forming a first complex in the cell in which the binding molecule binds to the ribosome-RNA complex;
  • binding molecule is shown in the following formula:
  • step (d) contacting the product obtained in step (c) with a first degradation reagent that degrades RNA other than ribosome-RNA complexes;
  • step (e) contacting the product obtained in step (d) with a carrier with streptavidin to form a second complex in which the carrier with streptavidin is combined with the first complex;
  • step (f) contacting the product obtained in step (e) with a second degradation reagent, the second degradation reagent degrades the protein in the second complex to obtain RNA after degrading the protein;
  • the RNA after the above-mentioned degraded protein is enriched.
  • the enriched RNA is eluted to remove proteins and/or RNA (eg, rRNA) other than mRNA. Methods for removing rRNA therein are known to those skilled in the art.
  • the enriched RNA is reverse transcribed into cDNA (eg, rRNA into ribosomal cDNA) by a kit.
  • the ribosomal cDNA in the reverse transcription product cDNA is removed by a kit.
  • the kit is a minilibrary kit (eg, Takara SMAETer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian-634413).
  • the present application provides a method for single-cell translational genome sequencing, the method comprising:
  • step (b) providing a binding molecule, contacting the binding molecule with the single cell obtained in step (a), forming in the cell a bond between the binding molecule and the ribosome-RNA complex in the single cell first complex;
  • binding molecule is shown in the following formula:
  • step (d) contacting the product obtained in step (c) with a first degradation reagent that degrades RNA other than ribosome-RNA complexes;
  • step (e) contacting the product obtained in step (d) with a carrier with streptavidin to form a second complex in which the carrier with streptavidin binds to the first complex;
  • step (f) contacting the product obtained in step (e) with a second degradation reagent, the second degradation reagent degrades the protein in the second complex to obtain RNA after degrading the protein;
  • step (g) constructing a library using the RNA obtained in step (f);
  • step (h) Sequencing the library obtained in step (g) to obtain sequencing information of the translation group.
  • the RNA enriched in step (f) is eluted to remove proteins and/or RNA (eg, rRNA) other than mRNA. Methods for removing rRNA therein are known to those skilled in the art.
  • the enriched RNA is reverse transcribed into cDNA (eg, rRNA into ribosomal cDNA) by a kit.
  • the ribosomal cDNA in the reverse transcription product cDNA is removed by a kit.
  • the kit is a minilibrary kit (eg, Takara SMAETer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian-634413).
  • the present application provides a method for simultaneously performing single-cell transcriptome sequencing and translation genome sequencing, the method comprising:
  • step (b) providing a binding molecule, contacting the binding molecule with the single cell obtained in step (a), forming in the cell a bond between the binding molecule and the ribosome-RNA complex in the single cell first complex;
  • binding molecule is shown in the following formula:
  • step (d) contacting the product obtained in step (c) with a first degradation reagent that degrades RNA other than ribosome-RNA complexes;
  • step (e) contacting the product obtained in step (d) with a carrier with streptavidin to form a second complex in which the carrier with streptavidin binds to the first complex;
  • step (g) contacting the product obtained in step (e) with a second degradation reagent, the protein in the second complex of the second degradation reagent, to obtain RNA after degrading the protein, i.e. the second RNA;
  • step (h) using the first RNA obtained in step (f) and the second RNA obtained in step (g) to establish libraries respectively;
  • step (i) Sequencing the libraries obtained in step (h) to obtain sequencing information of transcriptome and translation genome.
  • the RNA enriched in step (f) is eluted to remove proteins and/or RNA (eg, rRNA) other than mRNA.
  • RNA eg, rRNA
  • Methods for removing rRNA therein are known to those skilled in the art.
  • the enriched RNA is reverse-transcribed into cDNA (eg, Takara SMAETer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian-634413) by a kit (eg, a mini-library kit, eg, , reverse transcription of rRNA into ribosomal cDNA).
  • the ribosomal cDNA in the reverse transcription product cDNA is removed by a kit.
  • the library is constructed by a kit (e.g., a minilibrary kit, e.g., Takara SMAETer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian-634413).
  • a kit e.g., a minilibrary kit, e.g., Takara SMAETer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian-634413.
  • the ribosome-bound RNA is mRNA.
  • the RNA other than the ribosome-RNA complex comprises episomal mRNA, rRNA, tRNA, or any combination thereof.
  • the single cell is pretreated to allow ribosomes to lodge at one or more defined regions of the RNA molecule and form ribosome-RNA complexes.
  • ribosome-RNA complexes are formed in vivo/intracellularly.
  • the pretreatment is selected from the group consisting of: contacting the cells with cycloheximide, or contacting the cells with harringtonine, or freezing the cells in liquid nitrogen, or any combination.
  • the binding molecule enters the single cell, binds to said ribosome-RNA complex, and forms a first complex.
  • the binding molecule is contacted with the single cell obtained in step (a) for 15 seconds to 30 minutes, for example, for 15 seconds to 30 seconds, 30 seconds to 45 seconds, 45 seconds to 1 minute, 1 minute -2 minutes, 2 minutes-3 minutes, 3 minutes-4 minutes, 4 minutes-5 minutes, 5 minutes-10 minutes, 10 minutes-15 minutes, 15 minutes-20 minutes, 20 minutes-30 minutes and within the range any time period.
  • the binding molecule is contacted with the single cells obtained in step (a) for 1 minute to 30 minutes.
  • the binding molecule is contacted with the single cells obtained in step (a) for 30 seconds, 1 minute, 2 minutes, 5 minutes, 15 minutes or 30 minutes.
  • the concentration of the binding molecule is 50 to 1000 ⁇ M, for example, 50 ⁇ M-75 ⁇ M, 75 ⁇ M-100 ⁇ M, 100 ⁇ M-250 ⁇ M, 250 ⁇ M-500 ⁇ M, 500 ⁇ M-750 ⁇ M, 750 ⁇ M-1000 ⁇ M and any other within the range concentration.
  • the concentration of the binding molecule is 50 ⁇ M, 100 ⁇ M, 200 ⁇ M, 500 ⁇ M, 750 ⁇ M or 1000 ⁇ M.
  • the single cell is obtained from a source selected from a prokaryote, a eukaryote (eg, a mammal, eg, a human), a virus, or a viroid.
  • a source selected from a prokaryote, a eukaryote (eg, a mammal, eg, a human), a virus, or a viroid.
  • the single cell is obtained from a sample comprising a repertoire of RNA-ribosome complexes.
  • the samples include, but are not limited to, whole blood; blood products, such as plasma or serum; swabs, including but not limited to oral swabs, throat swabs, vaginal swabs, urethral swabs, cervical swabs, throat swabs, Rectal swab, lesion swab, abscess swab, nasopharyngeal swab, etc.; urine; sputum; saliva; semen; lymph fluid; amniotic fluid; cerebrospinal fluid; peritoneal effusion; pleural effusion; fluid from cyst; aspirates; lung lavage fluid; lung aspirates; tissues, including but not limited to, liver, spleen, kidney, lung, intestine, brain, heart, muscle, pancreas, cell culture, plant tissue or samples, And lysates, extract
  • the form of the cells includes directly harvested cells, as well as processed cells, such as preserved, fixed and/or stabilized cells.
  • the cells are in a form selected from: cultured cells, tissue isolated cells, frozen cells, paraffinized cells, or any combination thereof.
  • RNA-ribosome complexes can be released from single cells using well known chemical, physical or electrolytic lysis methods.
  • chemical methods typically use lysates to disrupt cells and extract RNA-ribosome complexes from them.
  • the lysing method comprises: mechanical disruption, sonication, contacting with a cell lysate, or any combination thereof.
  • the extracted RNA-ribosome complexes can be separated from other components of the crude extract (eg, denatured proteins, cell membrane particles, salts, etc.). Particulate matter is typically removed by centrifugation, filtration, flocculation, and the like.
  • nucleic acids not bound to ribosomes are degraded using a first degradation reagent.
  • the RNA with which the ribosome forms a ribosome-RNA complex is protected from degradation due to steric protection by the ribosome.
  • nucleic acid degradation treatments are known in the art, including thermal degradation, acid hydrolysis, and enzymatic digestion.
  • the first degrading agent is a nucleolytic enzyme.
  • the first degradation reagent comprises RNase and optionally DNase.
  • the first degradation reagent comprises ribonuclease and optionally DNase.
  • DNases include: endodeoxyribonucleases DNases, (eg, DNase I), exodeoxyribonucleases (eg, exodeoxyribonucleases I, III, 6, and 8), or any combination thereof.
  • ribonucleases include: endoribonucleases (for example, RNase A, H.III, L, P, PhyM, T1, T2U2 and V), exoribonucleases (for example, PNP enzyme, RNase PH, R, D, T, oligoribonuclease, exoribonuclease I and exoribonuclease II), or any combination thereof.
  • endoribonucleases for example, RNase A, H.III, L, P, PhyM, T1, T2U2 and V
  • exoribonucleases for example, PNP enzyme, RNase PH, R, D, T, oligoribonuclease, exoribonuclease I and exoribonuclease II
  • RNA when it is desired to degrade genomic DNA and RNA, a combination of one or more RNases and one or more DNases can be used. Nucleic acid degradation can be carried out with various enzyme amounts, incubation conditions, incubation temperatures, incubation buffers, incubation times and incubation lengths, and the specific conditions are well known to those skilled in the art.
  • the population of RNA fragments protected from nucleic acid degradation is purified prior to its use in downstream processing.
  • Suitable methods for nucleic acid purification are well known in the art and are commercially available (eg, RNA precipitation methods, silica gel column-based methods, gel purification methods, etc.).
  • a second degradation reagent is used to degrade proteins in the ribosome-RNA complex (eg, RPL4 protein, RPL3 protein, RPS3A protein, and RPS14 protein in ribosomes).
  • proteins in the ribosome-RNA complex eg, RPL4 protein, RPL3 protein, RPS3A protein, and RPS14 protein in ribosomes.
  • the second degradation reagent comprises a protease.
  • said second degradation reagent comprises proteinase K.
  • the carrier is a magnetic bead.
  • ribosome refers to the well-known ribonucleoprotein particle, which has small and large subunits, that translates RNA into protein during protein synthesis. According to the sequence defined by the template messenger RNA (mRNA), multiple amino acids are linked via peptide bonds to form proteins. In bacteria, these subunits have sedimentation coefficients of 30 and 50 and are therefore referred to as “30S” and “50S” subunits, respectively. In some eukaryotes, the sedimentation coefficient is 40 and 60.
  • ribosome-RNA complex refers to the complex formed by the association of an RNA being translated or to be translated with a ribosome.
  • the term “streptavidin” is a basic glycoprotein extracted from ovalbumin, which is heat resistant and resistant to the action of various proteolytic enzymes. It can be combined with biotin and has better stability after being combined with biotin.
  • the interaction between biotin and avidin is the strongest non-covalent interaction known so far, with an affinity constant (K) of 1015 mol/L.
  • K affinity constant
  • the combination of the two has good stability and specificity, and is not affected by reagent concentration, pH environment, or organic solvents such as protein denaturants.
  • the term “carrier with streptavidin” is also capable of binding biotin and has good stability.
  • transcriptome refers to the collection of all transcripts in a cell under certain physiological conditions. Typically, the transcriptome includes messenger RNA, ribosomal RNA, transfer RNA, and non-coding RNA. Transcriptome sequencing includes all mRNAs in a tissue or cell, whether they are translated or not.
  • translation set refers to the collection of mRNAs that are being translated in a cell under certain physiological conditions.
  • the method of the present application (1) can be applied in microcells and single cells, realizes single-cell translation genome sequencing, accurately captures the ribosome-RNA complexes being translated, and truly reflects The status of the mRNA being translated under the physiological conditions of the cell; (2) reduce the false positive of the sequencing result; (3) the translation group and the transcriptome can be sequenced simultaneously at the single cell level, realizing the organic unification of the translation group and the transcriptome.
  • Figure 1 shows the immunofluorescence imaging results of four ribosomal proteins (RPL4, RPL3, RPS3A and RPS14) after single cells were treated with 3P molecules.
  • CTRL is the control group.
  • Figure 2 shows the results of the single-cell translation panel.
  • Figure 2A shows the repeatability of single-cell translation signals
  • Figure 2B shows the repeatability of 50-cell translation signals
  • Figure 2C shows the similarity of single-cell and 50-cell translation signals
  • Figure 2D shows the overall visual display The enrichment of normalized reads in single cells and 50 cell libraries
  • Figure 2E shows the enrichment of normalized reads in single cells and 50 cell libraries for a single gene.
  • Figure 3 shows the number of genes obtained by translational and transcriptome sequencing in the same cell.
  • Figure 4 shows the detection results of the binding of 3P molecules to ribosomal subunits after cells were treated with different concentrations (0 ⁇ M (control), 50 ⁇ M, 100 ⁇ M, 200 ⁇ M, 500 ⁇ M and 1000 ⁇ M).
  • Pulldown is the ribosome-RNA complex sample captured by magnetic beads after 3P molecule treatment of cells
  • input is the sample before magnetic bead capture after 3P molecule treatment of cells.
  • Figure 5 shows the fluorescence results of mouse oocytes treated with 3P molecules at different times (0s (control), 30s, 1min, 5min, 15min and 30min).
  • Figure 6 shows the fluorescence results of 3P molecular treatment of zebrafish embryonic cells at different times (0s (control), 30s, 1min, 5min, 15min and 30min).
  • the transcriptome and translation genome of a single cell are simultaneously sequenced, and the following are the experimental steps:
  • 3P molecule binding molecule
  • the concentration of 3P used is 500 ⁇ M. Take 1mL of M2 cell operation solution and add 1 ⁇ L of 500mM 3P, mix well and place it in a cell culture incubator at 37°C to preheat. Take oocytes, wash with M2 operating solution 3 times, treat in 3P-M2 for 1 minute, and wash 3 times with M2 operating solution. Use a mouth pipette to aspirate a single cell with 6 ⁇ L of lysis buffer (20mM Tris-HCl, pH 7.4, 150mM NaCl, 5mM MgCl2, 1mM DTT, 1% Triton X-100, 200U/ml RNase inhibitor, 25U /ml DNase) in a low-adsorption 1.5mL centrifuge tube.
  • lysis buffer (20mM Tris-HCl, pH 7.4, 150mM NaCl, 5mM MgCl2, 1mM DTT, 1% Triton X-100, 200U/ml RNase inhibitor, 25U /ml
  • Immunofluorescence imaging detection of ribosomes In order to confirm that 3P molecules can enter cells and bind to ribosome-RNA complexes, a ribosomal subunit-specific antibody with a fluorescent group (Anti-RPL4 (purchased from Abclonal; Cat. No. A7620); Anti-RPL3 (available from Proteintech; Catalog No. II005-I-AP); Anti-RPS3A (available from Abclonal; A5885); Anti-RPS14 (available from Abclonal; Catalog No. A6727); and, Anti-rabbit (IgG) - FITC (purchased from Thermo; Cat. No. F0382) and Streptavidin-Cy3 (purchased from Sigma; Cat. No. S6402-1ML]), and detection of the fluorophore of the antibody by fluorescence microscopy.
  • Anti-RPL4 purchased from Abclonal; Cat. No. A7620
  • Anti-RPL3 available from Proteintech; Catalog No. II00
  • Dynabead MyOne magnetic beads purchased from Invitrogen, product number 65002
  • BW buffer 5mM Tris-HCl (pH 7.5), 500 ⁇ M EDTA, 1M NaCl, 0.05% TritonX-100
  • BW buffer 5mM Tris-HCl (pH 7.5), 500 ⁇ M EDTA, 1M NaCl, 0.05% TritonX-100
  • the blocked magnetic beads were resuspended with 194 ⁇ L of blocking buffer, mixed evenly and added to the fragmented cell lysate. Place the centrifuge tube on a rotating rack at 4°C and incubate for 1 hour or overnight.
  • PK buffer 100mM Tris-Cl (pH 7.5), 50mM NaCl, 10mM EDTA, 1% SDS, 190ug/mL Pronase K). Resuspend the magnetic beads with 200 ⁇ L PK buffer, mix thoroughly, place on a metal bath at 55°C, 1100rpm, and digest for 1 hour.
  • RNA extraction solution 200 ⁇ L RNA extraction solution to the digested centrifuge tube, mix thoroughly on a vortex shaker, and place on ice for 15 minutes for extraction. Place the centrifuge tube in a centrifuge at 4°C and centrifuge at 13300rpm for 15 minutes. After centrifugation, carefully take out the centrifuge tube and place it on the magnetic stand, pipette the supernatant (about 200 ⁇ L) into a new low adsorption 1.5mL centrifuge tube, and label Pulldown-RNA.
  • RNA extraction solution to Supernatant-RNA, mix thoroughly on a vortex shaker, and place on ice for 15 minutes for extraction. Place the centrifuge tube in a centrifuge at 4°C and centrifuge at 13300rpm for 15 minutes. After centrifugation, carefully take out the centrifuge tube and put it on the magnetic stand, pipette the supernatant (about 200 ⁇ L) into a new low adsorption 1.5mL centrifuge tube, and label Supernatant-RNA.
  • Pulldown-RNA was treated as fragmented RNA
  • Supernatant-RNA was treated as total RNA
  • scRibo-seq and scRNA-seq were performed according to the library construction instructions of the kit (Takara SMARTer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian-634413) library construction.
  • the successfully constructed library was subjected to paired-end sequencing through the Hiseq-PE150 sequencing platform.
  • the original reads obtained by sequencing were evaluated for data quality using FastQC software. Cutadapt software was used to remove adapter sequences, and Trimmomatic software removed low-quality bases with an error rate > 1% and shorter reads with a length ⁇ 35 nt.
  • the preprocessed paired-end reads were compared to the reference genome sequence of the mouse mm9 version using Bowtie2 software, using default parameters.
  • the aligned reads were calculated using HTSeq software for the number of reads corresponding to each gene (parameter: -m union-s no) and performed RPKM calculation (Reads per kilobase per million mapped reads). Translation efficiency was calculated by dividing the translation signal by the expression signal.
  • Figure 1 shows the immunofluorescence imaging results of single cells treated with 3P molecules.
  • the experimental results confirmed that 3P molecules entered single cells and combined with ribosome-RNA complexes.
  • This example intends to explore the concentration and time of binding molecules (hereinafter referred to as 3P molecules) to treat single cells, and the experiment is carried out according to the experimental procedures described in Example 1, only changing the concentration and/or time of 3P molecules to treat single cells.
  • 3P molecules concentration and time of binding molecules
  • 3P molecules were treated with mouse oocytes and zebrafish embryo cells at 500 ⁇ M concentration for 0s (control), 30s, 1min, 5min, 15min and 30min, respectively;
  • 3P molecules were treated with human HeLa cells at concentrations of 0 ⁇ M, 50 ⁇ M, 100 ⁇ M, 200 ⁇ M, 500 ⁇ M, 750 ⁇ M and 1000 ⁇ M for 1 min.
  • FIG. 4 shows the experimental results of Figure 4 to Figure 6.
  • the results in Figure 4 show that cells treated with 50 ⁇ M, 100 ⁇ M, 200 ⁇ M, 500 ⁇ M, 750 ⁇ M and 1000 ⁇ M 3P can enrich ribosomal subunits.
  • Figure 5 shows the fluorescence results of 3P molecule treatment of mouse oocytes for 0s (control), 30s, 1min, 5min, 15min and 30min.
  • Figure 6 shows the fluorescence results of zebrafish embryonic cells treated with 3P molecules at 0s (control), 30s, 1min, 5min, 15min and 30min.

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Abstract

La présente invention concerne un procédé pour obtenir un ARN lié à un ribosome dans une cellule unique. En outre, la présente invention concerne un procédé de séquençage du translatome unicellulaire, et un procédé permettant de réaliser simultanément le séquençage du transcriptome et le séquençage du translatome unicellulaires. Le procédé permet de capturer avec précision un complexe ribosome-ARN en cours de traduction, ce qui reflète véritablement l'état de l'ARNm en cours de traduction dans des conditions physiologiques cellulaires. En outre, le séquençage du translatome et le séquençage du transcriptome peuvent être réalisés simultanément au niveau d'une seule cellule, réalisant ainsi l'unité du translatome et du transcriptome.
PCT/CN2021/115194 2021-07-13 2021-08-30 Procédé de séquençage du ranslatome unicellulaire WO2023284076A1 (fr)

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