WO2023284076A1 - Single-cell translatome sequencing method - Google Patents

Abstract

The present invention relates to a method for obtaining a ribosome-bound RNA in a single cell. Further, the present invention relates to a single-cell translatome sequencing method, and a method for simultaneously performing single-cell transcriptome sequencing and translatome sequencing. The method can accurately capture a ribosome-RNA complex being translated, which truly reflects the state of mRNA being translated under cellular physiological conditions. In addition, translatome sequencing and transcriptome sequencing can be simultaneously performed at a single-cell level, achieving the unity of translatome and transcriptome.

Description

A method for single-cell translational genome sequencing technical field

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.

Background technique

The translation process of messenger RNA (mRNA) is the central link of gene expression, and translation regulation plays an important role in different physiological processes. Ribosome profiling (Ribo-seq) 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.

Traditional Ribo-seq technology is one of the basic methods to study translation regulation. However, this method has inherent disadvantages: (1) The initial amount of cells is large: the use of sucrose density gradient centrifugation to enrich ribosome complexes in cell or tissue lysates often requires an initial amount of more than one million cells, which cannot Reach micro, trace or even single-cell level, so this technology cannot be applied to the study of cell differences (2) High false positive: not all ribosome complexes obtained by sucrose density gradient centrifugation Ribosomes stuck on mRNA and some microsomes composed of nucleic acid and protein will also be enriched, resulting in inevitable bias in the results.

At present, some Ribo-seq technologies have also been developed, but these technologies still have some disadvantages. For example, 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 heterogeneity between cells has always been a problem that cannot be ignored in biological research. With the rapid development of various sequencing technologies, the emergence of single-cell sequencing technology has solved the problem of being balanced out by mixing a large number of initial cells. The issue of cellular heterogeneity. At present, single-cell transcriptome sequencing technology is becoming more and more mature, and single-cell western blotting and single-cell protein profiling techniques are widely used, but in terms of single-cell translational genome, some breakthrough technologies are needed to fill the gap.

Contents of the invention

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.

Therefore, in a first aspect, the application provides a method for obtaining RNA bound to ribosomes in a single cell, the method comprising:

(a) providing a single cell, and pretreating the single cell to form a ribosome-RNA complex;

(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;

Wherein, the binding molecule is shown in the following formula:

(c) the product obtained in cracking step (b);

(d) contacting the product obtained in step (c) with a first degradation reagent that degrades RNA other than ribosome-RNA complexes;

(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;

Optionally, centrifuging the second complex to enrich the second complex;

(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;

Optionally, the RNA after the above-mentioned degraded protein is enriched.

In certain embodiments, 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. In certain embodiments, the enriched RNA is reverse transcribed into cDNA (eg, rRNA into ribosomal cDNA) by a kit. In some embodiments, the ribosomal cDNA in the reverse transcription product cDNA is removed by a kit. In certain embodiments, the kit is a minilibrary kit (eg, Takara SMAETer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian-634413).

In a second aspect, the present application provides a method for single-cell translational genome sequencing, the method comprising:

(a) providing a single cell, and pretreating the single cell to form a ribosome-RNA complex;

(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;

Wherein, the binding molecule is shown in the following formula:

(c) the product obtained in cracking step (b);

(d) contacting the product obtained in step (c) with a first degradation reagent that degrades RNA other than ribosome-RNA complexes;

(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;

Optionally, centrifuging the second complex to enrich the second complex;

(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;

Optionally, enriching the RNA after the above degraded protein;

(g) constructing a library using the RNA obtained in step (f);

(h) Sequencing the library obtained in step (g) to obtain sequencing information of the translation group.

In certain embodiments, 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. In certain embodiments, the enriched RNA is reverse transcribed into cDNA (eg, rRNA into ribosomal cDNA) by a kit. In some embodiments, the ribosomal cDNA in the reverse transcription product cDNA is removed by a kit. In certain embodiments, the kit is a minilibrary kit (eg, Takara SMAETer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian-634413).

In a third aspect, the present application provides a method for simultaneously performing single-cell transcriptome sequencing and translation genome sequencing, the method comprising:

(a) providing a single cell, and pretreating the single cell to form a ribosome-RNA complex;

(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;

Wherein, the binding molecule is shown in the following formula:

(c) the product obtained in cracking step (b);

(d) contacting the product obtained in step (c) with a first degradation reagent that degrades RNA other than ribosome-RNA complexes;

(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;

(f) centrifuging the second complex to enrich the second complex; at the same time, collecting the supernatant and extracting the first RNA;

(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;

Optionally, enriching the above-mentioned second RNA;

(h) using the first RNA obtained in step (f) and the second RNA obtained in step (g) to establish libraries respectively;

(i) Sequencing the libraries obtained in step (h) to obtain sequencing information of transcriptome and translation genome.

In certain embodiments, 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. In certain embodiments, 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). In some embodiments, the ribosomal cDNA in the reverse transcription product cDNA is removed by a kit.

In certain embodiments, 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).

In certain embodiments, the ribosome-bound RNA is mRNA.

In certain embodiments, the RNA other than the ribosome-RNA complex comprises episomal mRNA, rRNA, tRNA, or any combination thereof.

In certain embodiments, 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. In certain embodiments, ribosome-RNA complexes are formed in vivo/intracellularly. In certain embodiments, 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.

In certain embodiments, the binding molecule enters the single cell, binds to said ribosome-RNA complex, and forms a first complex.

In certain embodiments, 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.

In certain embodiments, the binding molecule is contacted with the single cells obtained in step (a) for 1 minute to 30 minutes.

In certain embodiments, 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.

In some embodiments, the concentration of the binding molecule is 50 to 1000 μM, for example, 50 μM-75 μM, 75 μM-100 μM, 100 μM-250 μM, 250 μM-500 μM, 500 μM-750 μM, 750 μM-1000 μM and any other within the range concentration.

In certain embodiments, the concentration of the binding molecule is 50 μM, 100 μM, 200 μM, 500 μM, 750 μM or 1000 μM.

In certain embodiments, 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.

In certain embodiments, 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, extracts, or materials and components obtained from the above-mentioned samples, or any cells, microorganisms, viruses, etc. that may exist on or in the samples.

In certain embodiments, the form of the cells includes directly harvested cells, as well as processed cells, such as preserved, fixed and/or stabilized cells.

In certain embodiments, 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. For example, chemical methods typically use lysates to disrupt cells and extract RNA-ribosome complexes from them. In some embodiments, in step (c), the lysing method comprises: mechanical disruption, sonication, contacting with a cell lysate, or any combination thereof.

After extraction, 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.

In certain embodiments, nucleic acids not bound to ribosomes (eg, episomal mRNA, tRNA, rRNA, genomic DNA, etc.) are degraded using a first degradation reagent. In such embodiments, the RNA with which the ribosome forms a ribosome-RNA complex is protected from degradation due to steric protection by the ribosome.

Numerous nucleic acid degradation treatments are known in the art, including thermal degradation, acid hydrolysis, and enzymatic digestion.

In certain embodiments, the first degrading agent is a nucleolytic enzyme. In certain embodiments, the first degradation reagent comprises RNase and optionally DNase. In certain embodiments, the first degradation reagent comprises ribonuclease and optionally DNase.

In certain embodiments, DNases include: endodeoxyribonucleases DNases, (eg, DNase I), exodeoxyribonucleases (eg, exodeoxyribonucleases I, III, 6, and 8), or any combination thereof.

In some embodiments, 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.

For example, 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.

In certain embodiments, 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.).

In certain embodiments, 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). Many protein degradation treatments are known in the art, for example, enzymatic digestion and the like. In certain embodiments, the second degradation reagent comprises a protease. Preferably, said second degradation reagent comprises proteinase K.

In certain embodiments, the carrier is a magnetic bead.

Definition of Terms

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the operational steps of molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA used herein are all routine procedures widely used in the corresponding fields . Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "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.

As used herein, the term "ribosome-RNA complex" refers to the complex formed by the association of an RNA being translated or to be translated with a ribosome.

As used herein, 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. Moreover, 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. As mentioned above, the term "carrier with streptavidin" is also capable of binding biotin and has good stability.

As used herein, the term "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.

As used herein, the term "translation set" refers to the collection of mRNAs that are being translated in a cell under certain physiological conditions.

Beneficial Effects of the Invention

Compared with the prior art, 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.

Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are only for illustrating the present invention, rather than limiting the scope of the present invention. Various objects and advantages of this invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of the preferred embodiment.

Description of drawings

Figure 1 shows the immunofluorescence imaging results of four ribosomal proteins (RPL4, RPL3, RPS3A and RPS14) after single cells were treated with 3P molecules. Among them, CTRL is the control group.

Figure 2 shows the results of the single-cell translation panel. Among them, 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 μM (control), 50 μM, 100 μM, 200 μM, 500 μM and 1000 μM). Among them, Pulldown is the ribosome-RNA complex sample captured by magnetic beads after 3P molecule treatment of cells, and 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).

detailed description

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Unless otherwise indicated, the experiments and methods described in the examples were essentially performed according to conventional methods well known in the art and described in various references. For example, conventional techniques of molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention can be found in Sambrook, Fritsch and Maniatis ( Maniatis), MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F.M. Ausubel (F.M. Ausubel, et al., (1987)); METHODS IN ENZYMOLOGY series (Academic Publishing Company): PCR 2: A PRACTICAL APPROACH (M.J. McPherson (M.J. MacPherson, B.D. Hames and G.R. Taylor (eds. (1995)), and ANIMAL CELL CULTURE (ed. R.I. Freshney (1987)) .

In addition, those that do not indicate specific conditions in the examples are carried out according to conventional conditions or conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products. Those skilled in the art understand that the examples describe the present invention by way of example and are not intended to limit the scope of the claimed invention. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1. Sequencing of single-cell transcriptome and translation genome

In this embodiment, according to the method of the present application, the transcriptome and translation genome of a single cell are simultaneously sequenced, and the following are the experimental steps:

1. Prepare 500mM binding molecules (1000X)

Weigh 104.65g binding molecule (hereinafter referred to as 3P molecule) powder, dissolve it in 200μl DMSO, and store it in the dark at -20°C.

2. 3P treatment of single cells

The concentration of 3P used is 500μM. Take 1mL of M2 cell operation solution and add 1μL 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 μL 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.

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.

3. Cell Lysis

Place the centrifuge tube with lysis buffer on ice for 30 minutes, and flick the tube wall with your fingers every 5 minutes to fully lyse the cells.

4. RNA Fragmentation

Add 0.2 μL RNase I (purchased from Ambion, Cat. No. AM2295) to the cell lysate to make the final concentration about 3 U/μL, incubate on a rotating rack at room temperature, add 0.2 μL SuperRNase inhibitor (purchased from Ambion) after 30 minutes , Cat. No. AM2694), on ice.

5. Streptavidin Magnetic Bead Preparation

Take 2 μL of Dynabead MyOne magnetic beads (purchased from Invitrogen, product number 65002) in a 1.5 mL low-adsorption centrifuge tube, and use 50 μL of BW buffer (5mM Tris-HCl (pH 7.5), 500 μM EDTA, 1M NaCl, 0.05% TritonX-100 ) to wash the magnetic beads, flick the wall of the centrifuge tube, and let it stand for 2 minutes. Put the centrifuge tube on the magnetic stand, let it stand for 5 minutes to make the solution clear, suck off the supernatant, and complete a wash. Repeat wash 3 times.

Wash the magnetic beads once with 50 μL Bead-solution buffer A (50mM of NaCl, 100mM of NaOH, 0.1% TritonX-100), and then wash the magnetic beads once with 50 μL Bead-solution buffer B (100mM NaCl, 0.1% TritonX-100) .

Resuspend the magnetic beads with 50 μL blocking buffer (1% BSA, 20mM Tris-HCl (pH 7.4), 150mM NaCl, 5mM MgCl2, 1mM DTT, 1% Triton X-100), and place the magnetic beads in 4 °C on a rotating rack for 1 hour.

6. Capturing ribosome-RNA complexes with magnetic beads

The blocked magnetic beads were resuspended with 194 μL 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.

7. Obtain translation RNA (Pulldown-RNA) and supernatant RNA (Supernatant-RNA)

Remove the centrifuge tube from the spin rack, centrifuge briefly and place it on the magnetic stand for 5 minutes until the solution becomes clear. Transfer the supernatant to a new low-adsorption 1.5mL centrifuge tube and store at -20°C for subsequent Supernatant-RNA extraction and scRNA-seq library construction. The magnetic beads were washed 3 times with 100 μL washing buffer (150 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1% Triton X-100, 200 U/ml RNase inhibitor).

8. Proteinase K (Pronase K) digestion

Configure 200μL PK buffer (100mM Tris-Cl (pH 7.5), 50mM NaCl, 10mM EDTA, 1% SDS, 190ug/mL Pronase K). Resuspend the magnetic beads with 200μL PK buffer, mix thoroughly, place on a metal bath at 55°C, 1100rpm, and digest for 1 hour.

9. RNA extraction

Add 200 μL 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 μL) into a new low adsorption 1.5mL centrifuge tube, and label Pulldown-RNA.

Add 200 μL 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 μL) into a new low adsorption 1.5mL centrifuge tube, and label Supernatant-RNA.

Add 1 μL of glycogen, 1/10 volume (20 μL) of 3M sodium acetate and 3 times the volume (600 μL) of absolute ethanol to the Pulldown-RNA and Supernatant-RNA tubes, and place at -80°C for alcohol precipitation for 1 hour or overnight.

Take out the centrifuge tube after alcohol precipitation, centrifuge at 13300rpm in a centrifuge at 4°C for 30 minutes, and discard the supernatant. Add 1 mL of 75% ethanol to wash the precipitate, centrifuge at 13300 rpm for 10 minutes in a centrifuge at 4°C, and discard the supernatant. Then centrifuge at 13300 rpm for 2 minutes in a centrifuge at 4° C. to remove residual ethanol solution. Open the cap of the tube and place it in an ultra-clean workbench to dry for about 5-10 minutes. Dissolve the RNA pellet with 8 μL of water and store in a -80°C refrigerator.

10. RNA library construction

Pulldown-RNA was treated as fragmented RNA, Supernatant-RNA was treated as total RNA, and 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.

11. Sequencing and data analysis

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.

12. Experimental results

The processed products of the above experimental step 2 were detected by immunofluorescence microscopy. 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.

The sequencing results of the translation group (scRibo-Seq) and the transcriptome (scRNA-Seq) are shown in Table 1 and Figures 2-3. Rep1 and Rep2 are samples from two single cells, respectively. Among them, 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 derived from translational nuclear and transcriptome sequencing in the same cell. Experimental results prove that the present application has successfully established a method for obtaining single-cell translation groups. This method has high repeatability and stable results, and can be applied to a small amount of cells (for example, 50 cells) and a single cell. The method of the present application can simultaneously obtain the sequencing information of its translation group and transcript group in the same cell.

Table 1. Translational group (scRibo-Seq) and transcriptome (scRNA-Seq) sequencing results

Example 2. Concentration and time of binding molecules to treat single cells

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.

Specifically carry out the following experiments:

(1) 3P molecules were treated with mouse oocytes and zebrafish embryo cells at 500 μM concentration for 0s (control), 30s, 1min, 5min, 15min and 30min, respectively;

(2) 3P molecules were treated with human HeLa cells at concentrations of 0 μM, 50 μM, 100 μM, 200 μM, 500 μM, 750 μM and 1000 μM for 1 min.

The experimental results are shown in Figure 4 to Figure 6. The results in Figure 4 show that cells treated with 50 μM, 100 μM, 200 μM, 500 μM, 750 μM and 1000 μM 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. The experimental results confirmed that 500μM 3P treated cells can enter mouse cells and zebrafish embryo cells after 30s, and there is no significant difference between the treatment results of 1min and 5min, 15min, and 30min. The results show that 3P molecules and single Cells can enter the cells after 1min to 30min of contact.

Although the specific implementation of the present invention has been described in detail, those skilled in the art will understand that: according to all the teachings that have been published, various modifications and changes can be made to the details, and these changes are all within the protection scope of the present invention . The full scope of the invention is given by the claims appended hereto and any equivalents thereof.

Claims (10)

1. A method for obtaining RNA bound to ribosomes in a single cell, the method comprising:

   (a) providing a single cell, and pretreating the single cell to form a ribosome-RNA complex;

   (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;

   Wherein, the binding molecule is shown in the following formula:

   

   (c) the product obtained in cracking step (b);

   (d) contacting the product obtained in step (c) with a first degradation reagent that degrades RNA other than ribosome-RNA complexes;

   (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;

   Optionally, centrifuging the second complex to enrich the second complex;

   (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;

   Optionally, the RNA after the above-mentioned degraded protein is enriched.
2. A method for single-cell translational genome sequencing, the method comprising:

   (a) providing a single cell, and pretreating the single cell to form a ribosome-RNA complex;

   (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;

   Wherein, the binding molecule is shown in the following formula:

   

   (c) the product obtained in cracking step (b);

   (d) contacting the product obtained in step (c) with a first degradation reagent that degrades RNA other than ribosome-RNA complexes;

   (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;

   Optionally, centrifuging the second complex to enrich the second complex;

   (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;

   Optionally, enriching the RNA after the above degraded protein;

   (g) constructing a library using the RNA obtained in step (f);

   (h) Sequencing the library obtained in step (g) to obtain sequencing information of the translation group.
3. A method for simultaneously performing single-cell transcriptome sequencing and translation genome sequencing, the method comprising:

   (a) providing a single cell, and pretreating the single cell to form a ribosome-RNA complex;

   (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;

   Wherein, the binding molecule is shown in the following formula:

   

   (c) the product obtained in cracking step (b);

   (d) contacting the product obtained in step (c) with a first degradation reagent that degrades RNA other than ribosome-RNA complexes;

   (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;

   (f) centrifuging the second complex to enrich the second complex; at the same time, collecting the supernatant and extracting the first RNA;

   (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;

   Optionally, enriching the above-mentioned second RNA;

   (h) using the first RNA obtained in step (f) and the second RNA obtained in step (g) to establish libraries respectively;

   (i) Sequencing the libraries obtained in step (h) to obtain sequencing information of transcriptome and translation genome.
4. The method of claim 2 or 3, wherein the library is constructed by a kit (for example, a micro-library construction kit, for example, Takara SMAETer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian-634413).
5. The method of any one of claims 1-4, wherein the pretreatment is selected from the group consisting of: contacting the cells with cycloheximide, or contacting the cells with harringtonine, or contacting the cells Liquid nitrogen freezing, or any combination thereof.
6. The method according to any one of claims 1-5, wherein the binding molecule is in contact 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 -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 minutes and any time period within said range;

   Preferably, the binding molecule is contacted with the single cell obtained in step (a) for 1 minute to 30 minutes;

   Preferably, the binding molecules are contacted with the single cells obtained in step (a) for 30 seconds, 1 minute, 2 minutes, 5 minutes, 15 minutes or 30 minutes.
7. The method of any one of claims 1-6, wherein the concentration of the binding molecule is 50 to 1000 μM, for example, 50 μM-75 μM, 75 μM-100 μM, 100 μM-250 μM, 250 μM-500 μM, 500 μM-750 μM, 750 μM-1000 μM and Any concentration within the stated range;

   Preferably, the concentration of the binding molecule is 50 μM, 100 μM, 200 μM, 500 μM, 750 μM or 1000 μM.
8. The method of any one of claims 1-7, wherein the method has one or more features selected from the group consisting of:

   (1) the single cell is obtained from a source selected from the group consisting of prokaryotes, eukaryotes (e.g., mammals, e.g., humans), viruses or viroids;

   (2) The RNA bound to ribosomes is mRNA;

   (3) RNA other than the ribosome-RNA complex comprises free mRNA, rRNA, tRNA, or any combination thereof;

   (4) The form of the cells is selected from: cultured cells, cells isolated from tissues, frozen cells, paraffinized cells, or any combination thereof.
9. The method according to any one of claims 1-8, wherein, in step (c), the lysing method comprises: mechanical disruption, sonication, contact with cell lysate, or any combination thereof.
10. The method of any one of claims 1-9, wherein the method has one or more features selected from the group consisting of:

    (1) the first degradation reagent comprises RNase and optional DNase;

    Preferably, said first degradation reagent comprises ribonuclease and optionally DNase;

    Preferably, said ribonuclease comprises bovine pancreatic ribonuclease (RNase I) and optional DNase;

    (2) the second degradation reagent comprises protease; preferably, the second degradation reagent comprises proteinase K;

    (3) The carrier includes magnetic beads.

Images