Method for preparing non-ribosomal RNA-containing samples

Abstract

The present invention addresses the problem of providing a method for producing a non-ribosomal RNA-containing sample, said method comprising novel steps for removing ribosomes. According to the present invention, provided is a method for producing a non-ribosomal RNA-containing sample, said method comprising step (a) for, in a sample containing mRNA and ribosomes, releasing ribosomal subunits from mRNA, and step (b) for removing the ribosomal subunits released in step (a).

Description

[Technical field]

[0001] The present invention relates to a method for producing a non-ribosomal RNA-containing sample. The present invention relates to a method for analyzing non-ribosomal RNA using a non-ribosomal RNA-containing sample produced by the method for producing a non-ribosomal RNA-containing sample. Furthermore, the present invention relates to a kit used for carrying out the method for producing a non-ribosomal RNA-containing sample. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to Japanese Patent Application No. 2019-211933, filed November 25, 2019, the entire disclosure of which is expressly incorporated herein by reference. [Background technology]

[0002] With the advent of next-generation sequencers, the speed of sequence analysis has improved dramatically, making it possible to conduct research on large genomic regions. Next-generation sequencers can simultaneously process tens to hundreds of millions of randomly cut DNA fragments in parallel, and can obtain data from 1 billion bases (1 gigabase) to 100 billion bases (1 terabase) in a single sequencing run. In addition to whole-genome sequencing, next-generation sequencers are used in research on targeted sequencing, which targets genomic regions related to specific research subjects such as diseases, and epigenetics such as methylation sequencing.

[0003] Next-generation sequencers are also used to study the central dogma of molecular biology, namely the concept that genetic information is transmitted in the order of "DNA → (transcription) → mRNA (messenger RNA) → (translation) → protein." For example, RNA-Seq (RNA sequencing) can use next-generation sequencers to reveal the presence and amount of RNA in a biological sample at a specific moment, allowing comprehensive gene expression analysis. Furthermore, in research to clarify how the reaction of translation, in which proteins are made from mRNA, is controlled, a technique called ribosome profiling using next-generation sequencers can comprehensively analyze which codons of which mRNAs are being decoded by ribosomes, providing an overview of the state of translation.

[0004] Ribosome profiling is a technique based on deep sequencing of ribosome-protected mRNA fragments (Patent Document 1). Information from ribosome profiling can be useful for investigating translational regulation, measuring gene expression, measuring protein synthesis rates, or predicting protein abundance.

[0005] Patent Document 1: U.S. Patent No. 8,486,865

[0006] Non-patent document 1 Ingolia et al., 2009, Science, 324, 218-23 Non-patent document 2 Weinberg et al., 2016, Cell Rep, 14, 1787-1799 Non-patent document 3 McGlincy and Ingolia, 2017, Methods, 126, 112-129 The entire disclosures of Patent Document 1 and Non-Patent Documents 1 to 3 are incorporated herein by reference. Summary of the Invention [Problem to be solved by the invention]

[0007] Conventional ribosome profiling has a problem in that ribosomal RNA (rRNA) over-occupies the sequencing library, resulting in a low percentage of sequence reads available for analysis, specifically, the percentage of sequence reads that map to protein-coding regions (CDS). For example, Non-Patent Document 1 reports that when 42 million fragments obtained using a ribosome protection assay in the budding yeast Saccharomyces cerevisiae were sequenced, 7 million (16%) sequence reads were mapped to CDS, but most of the remaining were derived from rRNA. Since statistical analysis generally depends on the scale of mRNA sequence reads, a low yield of usable sequence reads in a library is an obstacle to analysis in deep sequencing-based approaches such as ribosome profiling.

[0008] In addition to ribosome profiling, RNA-seq also faces the problem of excessive rRNA contamination in the sequencing library. Non-Patent Document 2 reports that when an RNA-seq library was prepared from total RNA that was not enriched in mRNA, 90.2% of the 199.7 million reads were rRNA. The presence of excess rRNA sequence reads significantly reduces the efficiency of transcriptome analysis.

[0009] In order to reduce rRNA sequence reads, rRNA-subtraction oligonucleotides that can hybridize to rRNA and trap it on magnetic beads have been used (Non-Patent Documents 2, 3). However, even rRNA-depletion using rRNA-subtraction oligonucleotides could not sufficiently reduce rRNA sequence reads. Therefore, a new method for reducing rRNA sequence reads was required. Therefore, an object of the present invention is to provide a method for producing a non-ribosomal RNA-containing sample, which includes a new step for removing ribosomes. [Means for solving the problem]

[0010] As a result of intensive research aimed at solving the above problems, the present inventors have found that ribosomal subunits can be efficiently removed by peeling them off from mRNA. The present invention is based on this finding. According to the present invention, the following inventions are provided. [1] In a sample containing mRNA and ribosomes, (a) separating ribosomal subunits and mRNA; and and removing the ribosomal subunits detached in step (a). Method for producing non-ribosomal RNA-containing samples. [2] A method for producing a non-ribosomal RNA-containing sample according to [1], further comprising a step of degrading RNA or a step of fragmenting RNA in a sample containing mRNA and ribosomes. [3] The method for producing a non-ribosomal RNA-containing sample according to [1] or [2], wherein the step (a) of separating ribosomal subunits and mRNA is carried out using a chelating agent. [4] The method for producing a non-ribosomal RNA-containing sample according to any one of [1] to [3], wherein the step (b) of removing the ribosomal subunits detached in the step (a) is carried out by ultrafiltration. [5] A step of obtaining a non-ribosomal RNA-containing sample by carrying out the method for producing a non-ribosomal RNA-containing sample according to any one of [1] to [4]; and Determining the base sequence of RNA in a sample containing non-ribosomal RNA A method for analyzing non-ribosomal RNA, comprising: [6] A reagent for separating ribosomal subunits and mRNA, and means for removing ribosomal subunits, A kit used for carrying out the method for producing a non-ribosomal RNA-containing sample according to any one of [1] to [4]. [Brief description of the drawings]

[0011] [Figure 1]FIG. 1 is a schematic diagram of the present invention. In the conventional purification method (Purification (standard method) in the figure), after RNase treatment, a monosome fraction obtained by ultracentrifugation or gel-filtration is treated with a solution containing a protein denaturant (e.g., phenol, guanidine isothiocyanate) to purify a footprint. In the improved method of the present invention (Modified method in the figure), a monosome fraction obtained by ultracentrifugation or gel-filtration is treated with a chelating agent to separate ribosomes into large and small subunits and footprints (Ribosome splitting with chelating agent), and then the large and small subunits are removed (Removal of ribosome subunits) to purify the footprint. [Diagram 2] Figure 2 shows the number of reads obtained by analysis of libraries prepared by the standard method, the ribosome splitting method, the standard method + rRNA depletion, and the ribosome splitting method + rRNA depletion in the embodiment. "Mapped" means the number of reads mapped to the protein coding region (CDS), which corresponds to the number of ribosomes in the mRNA. RPM is reads per million. [Diagram 3] Figure 3 shows the Pearson correlation coefficient for the number of reads obtained in the analysis of libraries prepared by the standard method, the ribosome splitting method, the standard method + rRNA depletion, and the ribosome splitting method + rRNA depletion methods (each repeated twice) of the examples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The following description of the present invention may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "~" means a range including the numerical values ​​before and after "~" as the lower and upper limits. "nt" regarding the length of RNA means nucleotides.

[0013] RNA-seq is a technology that can profile gene expression levels genome-wide. In this specification, RNA-seq includes whole transcriptome sequencing (whole RNA-seq), which can comprehensively grasp the transcriptional profile of a cell at a biological moment, targeted RNA sequencing, which measures only the target transcript to analyze differential expression and allele-specific gene expression, and small non-coding RNA sequences (e.g., transfer RNA, snoRNA, snRNA, etc.) and microRNA sequences related to transcriptional and translational control. snRNA (small nuclear RNA) is a group of small RNAs present in the nucleus of eukaryotes, and is involved in various reaction processes including RNA splicing and rRNA processing together with other proteins. snoRNA (small nucleolar RNA) is a group of small RNAs involved in the chemical modification (methylation, pseudouridylation, etc.) of rRNA and other RNAs. MicroRNAs are classified as functional non-coding RNAs. They are functional nucleic acids that are encoded on the genome and undergo a multi-step production process to ultimately become small RNAs of 20 to 25 bases in length. They are involved in the regulation of basic biological phenomena such as cell development, differentiation, proliferation, and cell death.

[0014] Ribosome profiling is a technique that takes advantage of the fact that when an mRNA molecule is degraded by enzymes, the part of the mRNA to which ribosomes were bound remains protected from degradation. It determines the sequences of many parts of the mRNA to which ribosomes were bound, and determines the regions of mRNA that were actively being translated within a cell at a particular time.

[0015] As used herein, the term "footprint" refers to a portion of mRNA that remains protected from degradation by enzymes, etc. in ribosome profiling. The length of a footprint is about 40 nt or less, generally about 30 nt.

[0016] As used herein, a "read" or "sequence read" generally refers to a data string of A, T, C, and G bases determined from a DNA or RNA sample. A read or sequence read herein is specifically a sequence determined for a DNA fragment in a sequencing library prepared from a non-ribosomal RNA-containing sample of the invention.

[0017] As used herein, "non-coding RNA" is used as a general term for RNA that does not code for proteins, and examples of non-coding RNA include rRNA, transfer RNA (tRNA), mitochondrial ribosomal RNA (Mt-rRNA), mitochondrial transfer RNA (Mt-tRNA), chloroplast-derived ribosomal RNA, chloroplast-derived transfer RNA, snRNA, snoRNA, microRNA, etc. As used herein, "non-ribosomal RNA" is used as a general term for RNA other than ribosomal RNA (rRNA). Examples of non-ribosomal RNA include mRNA, transfer RNA (tRNA), mitochondrial-derived transfer RNA (Mt-tRNA), chloroplast-derived transfer RNA, snRNA, snoRNA, microRNA, etc.

[0018] <Method of producing a sample containing non-ribosomal RNA> The method for producing a non-ribosomal RNA-containing sample of the present invention comprises the steps of: (a) separating ribosomal subunits and mRNA; and The method includes a step (b) of removing the ribosomal subunits detached in the step (a).

[0019] Non-Patent Document 1 reported that 16% of the sequence reads obtained by ribosome profiling were mapped to CDS, while most of the rest were derived from rRNA. Non-Patent Document 2 reported that 90.2% of the sequence reads obtained when preparing an RNA-seq library from total RNA in which mRNA was not enriched were rRNA. Also in the Examples described below, it has been shown that 92% of all reads were derived from rRNA by the conventional standard method. To reduce sequence reads derived from rRNA, rRNA-subtraction oligonucleotides that can hybridize to rRNA and be trapped by magnetic beads have been used (Non-Patent Documents 2 and 3). In the Examples described below, it has been shown that even when this method was adopted, 77% of all reads were derived from rRNA and the reads of mRNA were 18%.

[0020] In the analysis of a library for ribosome profiling prepared from a non-ribosomal RNA-containing sample produced by the present invention, 23% of all reads were derived from mRNA, and it has been shown in the Examples described below that when combined with the method using rRNA-subtraction oligonucleotides, the reads of mRNA increased to 50% of all reads. According to the present invention, a non-ribosomal RNA-containing sample with a reduced rRNA content ratio can be produced. Therefore, if a library for ribosome profiling or RNA-seq is prepared from the non-ribosomal RNA-containing sample produced by the present invention, reads of mRNA, non-coding RNA, particularly small non-coding RNA and microRNA, can be efficiently obtained. Note that the non-ribosomal RNA-containing sample produced by the present invention is not limited to use in ribosome profiling and is also effective for RNA-seq.

[0021] <Sample containing mRNA and ribosome〉 The "sample containing mRNA and ribosomes" of the present invention means a crude extract (hereinafter referred to as lysate) of cells or tissues obtained by dissolving or disrupting a single cell, a cell population, a cultured cell, or a tissue containing mRNA and ribosomes, and the single cell, the cell population, the cultured cell, or the tissue is derived from any organism. Specific examples include, but are not limited to, lysates of bacteria, fungi, animal cells or tissues, plant cells or tissues, or cultured cells thereof. Lysates can be prepared by cell lysis using a surfactant or physical disruption (e.g., mechanical disruption, homogenization in a solution, sonication, freeze-thawing, disruption with a mortar and pestle), and the preparation method can be appropriately selected depending on the type of organism or cell or tissue. The method for producing a non-ribosomal RNA-containing sample of the present invention can include a step of dissolving or disrupting cells to obtain a lysate as a pretreatment.

[0022] Lysates are preferably prepared by gentle means, such as cell lysis using detergents, to avoid degradation or damage of ribosomes. For the same reason, protein denaturants and Mg 2+ It is preferable to prepare the lysate without using a chelating agent or an organic solvent such as phenol or chloroform. Since DNA interferes with the subsequent synthesis of cDNA, it may be decomposed using, for example, DNase. Furthermore, the lysate can be obtained in the presence of cycloheximide, a protein translation inhibitor. In one embodiment, the lysate can be obtained as a supernatant obtained by suspending cells in a buffer containing a surfactant and cycloheximide, incubating in the presence of DNase I (RNase-free), and centrifuging, as in the Examples described below.

[0023] A sample containing mRNA and ribosomes can be said to be a sample that may contain, in addition to mRNA and ribosomes, non-coding RNA, such as rRNA, transfer RNA (tRNA), mitochondrial ribosomal RNA (Mt-rRNA), mitochondrial transfer RNA (Mt-tRNA), chloroplast-derived ribosomal RNA, chloroplast-derived transfer RNA, snRNA, snoRNA, microRNA, and the like.

[0024] <Samples containing non-ribosomal RNA> The "non-ribosomal RNA-containing sample" of the present invention is obtained by carrying out a step (a) of peeling off ribosomal subunits and mRNA in a sample containing mRNA and ribosomes, and a step (b) of removing the ribosomal subunits peeled off in the step (a). The non-ribosomal RNA-containing sample may contain mRNA, transfer RNA (tRNA), mitochondrial-derived transfer RNA (Mt-tRNA), chloroplast-derived transfer RNA, snRNA, snoRNA, microRNA, etc., and may further be enriched for a specific RNA species. The non-ribosomal RNA-containing sample may be a sample for preparing a sequencing library, and the sequencing library may be, for example, a library for ribosome profiling or RNA-seq, but is not limited thereto. The rRNA content in the non-ribosomal RNA-containing sample of the present invention is reduced compared to the same content in the non-ribosomal RNA-containing sample obtained by a conventional method (e.g., the standard method of Example 1). The ratio of the number of rRNA reads determined in a sequencing library prepared from a non-ribosomal RNA-containing sample of the present invention to the total number of reads is reduced compared to the same ratio in a sequencing library prepared from a sample obtained by a conventional method (e.g., the standard method of Example 1).

[0025] <Step (a) of separating ribosomal subunits from mRNA> Ribosomes are huge RNA-protein complexes consisting of several rRNA molecules and about 50 kinds of proteins, and are composed of two particles, large and small, as a whole. In this specification, the two particles of the ribosome are called the large subunit and the small subunit, respectively. The specific structure of the ribosome, for example, in the ribosomes of prokaryotes, the large subunit and the small subunit are called the 50S subunit and the 30S subunit, respectively. The 50S subunit consists of 23S rRNA (2904nt), 5S rRNA (120nt) and 34 kinds of proteins, and has a molecular weight of 1.6 million, and the 30S subunit consists of 16S rRNA (1542nt) and 21 kinds of proteins, and has a molecular weight of 900,000. The assembly of these two subunits becomes the 70S particle, and its molecular weight is 2.7 million. In eukaryotic ribosomes, the large and small subunits are called the 60S and 40S subunits, respectively. The 60S subunit consists of 28S rRNA (4718nt), 5.8S rRNA (160nt), 5S rRNA (120nt) and 50 types of proteins, with a molecular weight of 3 million. The 40S subunit consists of 18S rRNA (1874nt) and 33 types of proteins, with a molecular weight of 1.5 million. The assembly of these two subunits forms the 80S particle.

[0026] Ribosomes are the site of translation, where mRNA is loaded, read, and converted into proteins. By separating the assembly of both subunits into large and small subunits, the loaded mRNA can be separated from the ribosome.

[0027] The step (a) of separating the ribosomal subunits from the mRNA can be carried out by any method. For example, the method can be carried out using Mg, which is necessary to maintain the association of the two subunits. 2+ This can be achieved by removing the Mg ions. 2+Ions can be removed by any method, for example, by a chelating agent. That is, the step (a) of peeling off the ribosome subunits and the mRNA can be carried out using a chelating agent. Examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), glycoletherdiaminetetraacetic acid (EGTA, GEDTA), etc., and ethylenediaminetetraacetic acid (EDTA) is particularly preferred. In the case of ethylenediaminetetraacetic acid (EDTA), the concentration of the chelating agent can be 0.1 mM to 30 mM, and preferably 5 mM to 15 mM. In the case of EDTA, the treatment with the chelating agent can be performed by placing the sample on ice for 30 seconds to 60 minutes, and the treatment time can be appropriately changed.

[0028] <Step (b) of removing the ribosome subunits detached in step (a)> The step (b) of removing the ribosomal subunits detached in the step (a) is a step of removing the large and small subunits detached in the step (a) of detaching the ribosomal subunits and mRNA from a sample containing mRNA and ribosomes. The removal of the large and small subunits can be carried out by adopting any method that can remove them based on their size, for example, ultrafiltration, size exclusion chromatography (SEC), etc. As described below, the molecular weight of the small subunit of prokaryotes, which is the smallest ribosomal subunit, is about 900,000. Transfer RNA (tRNA), mitochondrial transfer RNA (Mt-tRNA), chloroplast-derived transfer RNA, snRNA, snoRNA, and microRNA are sufficiently smaller than the smallest prokaryotic subunit of the ribosomal subunits, so that they can be separated from the large and small subunits by the above-mentioned method based on the difference in size. On the other hand, the length of mRNA varies, and in the case of humans, many of them exceed 1,000 bases. Therefore, it may not be possible to separate the large and small subunits due to differences in size. Therefore, it is preferable to carry out the step of fragmenting RNA described below prior to "step (b) of removing the ribosomal subunits detached in step (a)" and fragment the mRNA to a size that can be separated from the large and small subunits.

[0029] Ultrafiltration is a method of concentrating or removing components in a solution by passing the solution through a membrane. The molecular weight cut off (MWCO) is set for the ultrafiltration membrane, and molecules larger than the molecular weight cut off of the membrane are retained by the membrane, and molecules larger than the molecular weight cut off of the membrane are removed from the permeate. In the present invention, the step of removing the large and small subunits of ribosomes can be performed by ultrafiltration. More specifically, ultrafiltration is used to permeate RNA in a sample containing mRNA and ribosomes, and the large and small subunits of ribosomes are retained on the membrane, thereby obtaining a permeate containing RNA. The RNA permeated through the ultrafiltration membrane and recovered may be any RNA, such as mRNA, tRNA, Mt-tRNA, chloroplast-derived transfer RNA, snRNA, snoRNA, microRNA, etc., and rRNA may also be mixed in.

[0030] As the ultrafiltration membrane, an ultrafiltration membrane that can permeate RNA and retain the large and small subunits of ribosomes on the membrane can be used. The permeability and retention of ultrafiltration membranes vary depending on various conditions, such as filtration pressure, the presence of other solutes, molecular shape, adsorptivity, ionic strength, etc., so examples of ultrafiltration membrane selection are shown below, but are not limited to these, and an optimal membrane can be selected appropriately taking into consideration the RNA recovery rate and the speed of filtration processing.

[0031] The ultrafiltration membrane that can be used in step (b) of the method for producing a non-ribosomal RNA-containing sample of the present invention has a molecular weight cutoff (MWCO) of 10K (hereinafter, K is 10 3(representing the above), to 2000K, 10K to 1500K, 10K to 1000K, 10K to 900K, 10K to 800K, 10K to 700K, 10K to 600K, 10K to 500K, 10K to 400K, 10K to 300K, 10K to 100K, 30K to 2000K, 30K to 1500K, 30K to 1000K, 30K to 900K, 30K to 800K, 30K to 700K, 30K to 600K, 30K to 500K, 30K to 400K, 30K to 300K, or 30K to 100K.

[0032] More specifically, regarding the removal of large and small subunits by ultrafiltration membrane, since the molecular weight of the small subunit of prokaryote, which is the smallest among ribosomal subunits, is about 900,000, if an ultrafiltration membrane with a molecular weight cutoff (MWCO) of less than 900K, preferably an ultrafiltration membrane with a MWCO of 150K to 300K is used, the large and small subunits of prokaryote can be retained on the membrane and removed. Also, since the molecular weight of the small subunit of eukaryote is about 1.5 million, if an ultrafiltration membrane with a MWCO of less than 1500K, preferably an ultrafiltration membrane with a MWCO of 250K to 500K is used, the large and small subunits of eukaryote can be retained on the membrane and removed. From the viewpoint of the retention rate of large and small subunits on the membrane, it is preferable to use a membrane with a MWCO of 500K or less, and it is more preferable to use a membrane with a MWCO of 300K or less.

[0033] The method for producing a non-ribosomal RNA-containing sample of the present invention may include a step of decomposing or fragmenting RNA, as described below. When these steps are included, the RNA is decomposed so that the RNA remains as a footprint of 40 nt or less in length, or is fragmented to a length depending on the sequencing platform. Based on the molecular weight of the RNA (about 320.5 / base), the selection of an ultrafiltration membrane for recovering RNA fragments is considered below. For example, if a membrane with a MWCO in the range of 30K to 500K is used, it is possible to permeate at least about 40 nt RNA (molecular weight about 13000) and retain large and small subunits of prokaryotes and eukaryotes on the membrane. In addition, if an ultrafiltration membrane with a MWCO in the range of 100K to 500K is used, it is possible to permeate at least about 100 nt RNA (molecular weight about 32000) and retain large and small subunits of prokaryotes and eukaryotes on the membrane. If an ultrafiltration membrane with a MWCO in the range of 300K to 500K is used, at least about 500nt RNA (molecular weight of about 160,000) can be permeated, and the large and small subunits of prokaryotes and eukaryotes can be retained on the membrane. If an ultrafiltration membrane with a MWCO of 500K is used, at least about 1000nt RNA (molecular weight of about 320,000) can be permeated, and the large and small subunits of prokaryotes and eukaryotes can be retained on the membrane. It is considered that linear molecules such as RNA may pass through membranes that can block globular molecules of the same molecular weight.

[0034] In one embodiment, ultrafiltration can be performed using a centrifugal ultrafiltration filter unit, which is composed of a tube with an ultrafiltration membrane. The centrifugal ultrafiltration filter unit can be further inserted into a tube to form a double-layered centrifugal ultrafiltration tube. The centrifugal ultrafiltration filter unit can be used according to the manufacturer's instructions.

[0035] Step (b) of removing the ribosomal subunits detached in step (a) can be carried out by size exclusion chromatography. In one embodiment, step (b) of removing the ribosomal subunits detached in step (a) can be carried out by gel filtration chromatography using a spin column. Specifically, for example, as shown in Illustra TM MicroSpin TM This can be done using S-400 HR Columns (GE Healthcare, cat. no. 27-5140-01) as follows: 1. Mix the S-400 column thoroughly, remove the tip of the column, and place it in a 2 mL reservoir tube. 2. Add 700 μl of lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA , 1. Load 1 mM dithiothreitol (DTT), 100 μg / mL cyclohexamide, RNase-free water; keep on ice) onto the S-400 column and centrifuge at 740g, 4°C for 1 minute. Place the column into a new 2 mL reservoir tube. Repeat this process a total of three times. 3. Place the S-400 column into a new clean 1.5 mL tube. Load 100 μL of the solution obtained by carrying out the above "Step (a) of stripping ribosomal subunits and mRNA" onto the center of the resin in the column and centrifuge at 740g, 4°C for 2 minutes. 4. Load an additional 100 μL of lysate and centrifuge at 740g, 4°C for 2 minutes. Obtain the eluate.

[0036] In another embodiment, step (b) of removing the ribosomal subunits detached in step (a) can be performed by size exclusion chromatography using ultra-high pressure liquid chromatography (uHPLC) (Yoshikawa et al. eLife 2018;7:e36530 DOI: 10.7554 / eLife.36530). Specifically, a 7.8×300 mm column with 5 μm particles, such as a Thermo BioBasic SEC 300A, 1,000A, 2,000A column, or an Agilent Bio SEC-5 2,000A column, can be used. Using a Dionex Ultimate 3,000 Bio-RS uHPLC system (Thermo Fisher Scientific), equilibrate each SEC column with 2 column volumes (CV) of filtered SEC buffer (20 mM Hepes-NaOH (pH 7.4), 60 mM NaCl, 30 mM EDTA, 0.3% CHAPS, 0.2 mg / mL heparin, 2.5 mM DTT), inject 100 μL of 10 mg / mL filtered bovine serum albumin (BSA) solution diluted with PBS to block nonspecific interaction sites once, inject a standard solution containing 10 μL of 10 mg / mL BSA solution and 25 μL of HyperLadder 1 kb (BIOLINE) to monitor the column status, and then inject the solution obtained by carrying out the above-mentioned "step (a) of peeling off ribosomal subunits and mRNA" into the column. Chromatograms are monitored by measuring UV absorbance at 215, 260, and 280 nm with a data collection rate of 1 Hz by a diode array detector. The flow rate is 0.8 mL / min, and 48 × 100 μL fractions, 24 × 200 μL fractions, or 16 × 300 μL fractions are collected from 9 min to 14.6 min at 4 °C using 1 mL low protein binding 96 deep-well plates (Eppendorf). Peaks are quantified using a Chromeleon 6.8 chromatography data system (Thermo Fisher Scientific).

[0037] The sample after removing ribosomal subunits by ultrafiltration, size exclusion chromatography, etc. can be subjected to purification. Purification can be performed using a known RNA purification method, for example, using a solution containing phenol / chloroform, phenol and guanidine isothiocyanate, etc.

[0038] The method for producing a non-ribosomal RNA-containing sample of the present invention can be combined with known rRNA removal methods, monosome enrichment methods, and mRNA enrichment methods. Known rRNA removal methods that can be combined include a method using rRNA-subtraction oligonucleotides that can hybridize to rRNA and trap it on magnetic beads (Non-Patent Documents 2 and 3), which can be carried out using the Ribo-Zero (registered trademark) rRNA Removal Kit (Illumina). Also, known mRNA enrichment methods that can be combined include the polyA selection method, in which polyA-tailed RNA is selected from the oligonucleotides using Oligotex (registered trademark)-dT30 <super>It can be concentrated using an mRNA Purification Kit (Takara) or oligo(dT)-Dynabeads (registered trademark). Known methods for concentrating combinable monosomes include sucrose density gradient centrifugation, sucrose cushion centrifugation, gel filtration chromatography using the above spin column, and the like.

[0039] If a sequencing library is prepared from the non-ribosomal RNA-containing sample produced by the present invention, the number of reads of rRNA relative to the total number of reads can be reduced. Specifically, the ratio of the number of reads of rRNA to the total number of reads can be reduced to 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less. Furthermore, when the production method of the present invention is combined with a method using an rRNA-subtraction oligonucleotide that hybridizes to rRNA and can be trapped by magnetic beads (Non-Patent Documents 2 and 3), the number of reads of rRNA relative to the total number of reads can be further reduced. Specifically, the ratio of the number of reads of rRNA to the total number of reads can be reduced to 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, or 10% or less.

[0040] <Step of degrading or fragmenting RNA> The method for producing a non-ribosomal RNA-containing sample of the present invention can further include a step of degrading or fragmenting RNA in a sample containing mRNA and ribosomes. Ribosome profiling usually includes a step of degrading RNA. In the present invention, the step of degrading RNA can be carried out before the step (a) of separating the ribosome subunits from mRNA. Also, when the RNA of interest contained in the non-ribosomal RNA-containing sample is long and cannot be separated based on the size of the large and small subunits, fragmentation of mRNA can be carried out before the step (b) of removing the ribosome subunits separated in step (a).

[0041] The step of degrading RNA can be carried out for the purpose of obtaining a footprint to be analyzed, for example, by ribosome profiling. The step of degrading RNA can be carried out, for example, by enzymatic degradation. RNA degradation refers to modifying RNA so that it is shorter in length than before degradation, and enzymatic degradation of RNA refers to degrading RNA using an enzyme that can modify RNA so that it is shorter in length than before degradation. The enzyme used may be a ribonuclease (RNA degrading enzyme) such as an endoribonuclease or an exoribonuclease, and a single-strand specific RNA endonuclease, for example, RNase I, can be used. Other enzymes that can be used for RNA degradation include RNase A, RNase T1, etc. RNA degradation may be carried out by partial alkaline hydrolysis, which will be described later.

[0042] In one embodiment, in the method for producing a non-ribosomal RNA-containing sample for preparing a library for ribosome profiling, the step of degrading RNA is carried out before the step (a) of peeling off ribosomal subunits and mRNA, and the RNA degradation can be carried out by RNase I digestion. By RNase I digestion, RNA molecules in a sample containing mRNA and ribosomes are degraded, but a part of the mRNA to which a ribosome is bound is protected from degradation. A part of the mRNA to which one ribosome is bound is called a monosome. The monosome in a sample containing mRNA and ribosomes can be concentrated by sucrose density gradient centrifugation, sucrose cushion centrifugation, gel filtration chromatography using the above-mentioned spin column, or the like.

[0043] The step of fragmenting the RNA can be carried out, for example, in step (b) of removing the ribosomal subunits detached in step (a) above, in order to make the RNA small enough so that the ribosomal subunits can be separated on the basis of size, and can be performed by any means capable of achieving this to the desired size.

[0044] The step of fragmenting RNA can be carried out, for example, by enzymatic fragmentation, chemical fragmentation, and / or mechanical fragmentation. RNA fragmentation refers to cutting RNA into appropriate fragment sizes, for example, cutting into lengths according to the sequencing platform. In one embodiment, RNA fragmentation can be carried out by partial alkaline hydrolysis. Partial alkaline hydrolysis can be carried out, for example, by adding 10 μL of 2x alkaline hydrolysis solution (2 mM EDTA, 12 mM Na2CO3, 88 mM NaHCO3, pH 9.3) to an equal amount of RNA-containing solution (e.g., lysate) and mixing, incubating the mixture at 95°C for 20 minutes, placing on ice after incubation, and adding 300 μL of 0.3 M NaOAc (pH 5.2). Fragmentation by partial alkaline hydrolysis can be performed with high degree of control so that the RNA is of an appropriate size, but for the purposes of the present invention, the RNA is fragmented to, for example, 100 to 3000 nt, preferably 100 to 1000 nt, more preferably 100 to 500 nt, and even more preferably 100 to 300 nt.

[0045] In one embodiment, RNA fragmentation can be performed by ultrasonic shearing. For example, ultrasonic shearing can be performed by placing the lysate in a 15 mL Falcon tube and sonicating it in a water bath at 4°C using an existing ultrasonic crusher. Fragmentation by ultrasonication can be performed with high control so that the RNA has an appropriate size, but for the present invention, the RNA is fragmented to, for example, 100 to 3000 nt, preferably 100 to 1000 nt, more preferably 100 to 500 nt, and even more preferably 100 to 300 nt.

[0046] In one embodiment, RNA fragmentation can be carried out enzymatically. For example, it is preferable to use an enzyme that can fragment single-stranded RNA randomly and without bias to a desired size, regardless of the base sequence. Specifically, RNase I, RNase A, RNase T1, RNase T2, MNase (Micrococcal Nuclease), RNase V1, RNase S1, etc. can be used. Enzymatic fragmentation can be carried out with high control so that RNA has an appropriate size, but for the present invention, it is fragmented to, for example, 100 to 3000 nt, preferably 100 to 1000 nt, more preferably 100 to 500 nt, and even more preferably 100 to 300 nt.

[0047] In the method for producing a non-ribosomal RNA-containing sample for preparing an RNA-seq library, the step of fragmenting RNA and the step (a) of separating ribosomal subunits and mRNA can be performed in any order, as long as the ribosomes are not destroyed.

[0048] In yet another embodiment, the method for producing a non-ribosomal RNA-containing sample may be carried out without carrying out the step of decomposing or fragmenting RNA, and a non-ribosomal RNA-containing sample may be produced. For example, this is the case when preparing a library for RNA-seq for analyzing small non-coding RNA (e.g., tRNA, snoRNA, snRNA, etc.) or microRNA. Small non-coding RNA is short, about 200 nt or less in length, and microRNA is short, about 30 nt or less in length, and can be separated from ribosomal subunits based on size without fragmentation.

[0049] <Method of analyzing non-ribosomal RNA> The method for analyzing non-ribosomal RNA of the present invention includes a step of obtaining a non-ribosomal RNA-containing sample by carrying out the above-mentioned method for producing a non-ribosomal RNA-containing sample, and a step of determining the base sequence of RNA in the non-ribosomal RNA-containing sample. Non-ribosomal RNA includes mRNA (including footprint), tRNA, Mt-tRNA, chloroplast-derived transfer RNA, snRNA, snoRNA, microRNA, etc. More specifically, the method for analyzing non-ribosomal RNA means an RNA-seq (RNA sequencing) method or a ribosome profiling method.

[0050] The step of determining the base sequence of RNA in a non-ribosomal RNA-containing sample is a step of determining the base sequence of RNA using a non-ribosomal RNA-containing sample obtained by carrying out a method for producing a non-ribosomal RNA-containing sample, and the determination of the base sequence includes the determination of the bases constituting the RNA as well as the determination of chemical modifications in the bases constituting the RNA. The determination of the base sequence of RNA may be carried out using an amplification product contained in a sequencing library prepared from the non-ribosomal RNA-containing sample.

[0051] A method for preparing a sequencing library typically includes a step of reverse transcribing a cDNA using a reverse transcriptase and amplifying the obtained reverse transcription product using an appropriate nucleic acid amplification method. Here, "amplifying (a nucleic acid)" refers to a process of subjecting the nucleic acid to at least one round of extension, replication, or transcription for the purpose of increasing (e.g., exponentially increasing) the number of copies of the nucleic acid. The copy of the nucleic acid may be a complementary copy of the nucleic acid. In addition, in this process, it is more preferable to perform multiple rounds of extension, replication, or transcription. The nucleic acid amplification method is not particularly limited, and examples thereof include PCR amplification and rolling circle amplification. In addition, the above-mentioned Patent Document 1 and Non-Patent Documents 1 to 3 can also be appropriately referred to for the method for preparing a sequencing library.

[0052] In one embodiment, for the preparation of a sequencing library for ribosome profiling, the method for producing a non-ribosomal RNA-containing sample of the present invention is carried out to obtain a non-ribosomal RNA-containing sample that selectively contains footprints.For example, in a sample containing mRNA and ribosomes (a sample in which a lysate is obtained in the presence of cycloheximide, RNA is decomposed with RNase I, and monosomes are enriched by carrying out the sucrose cushion method), a method for producing a non-ribosomal RNA-containing sample is carried out, comprising a step (a) of peeling off ribosomal subunits and mRNA, and a step (b) of removing the ribosomal subunits peeled off in step (a), to obtain a non-ribosomal RNA-containing sample.Thereby, a non-ribosomal RNA-containing sample that selectively contains footprints at the time of preparing the lysate can be obtained. The preparation of a sequencing library for ribosome profiling may be carried out as described in the Examples below, by subjecting a non-ribosomal RNA-containing sample to denaturing polyacrylamide gel electrophoresis together with an RNA size marker, excising RNA of 26 nt to 34 nt length and purifying it from the gel, and then providing a linker, reverse transcribing it into cDNA, circularizing it, amplifying it by PCR, and adding a barcode according to the Examples below. The addition of a linker, adapter, or barcode, reverse transcription, and PCR amplification reaction after excising RNA of the desired length and purifying it from the gel can be carried out according to any known method, preferably any known method used in analysis with a next-generation sequencer.

[0053] In one embodiment, for the preparation of a sequencing library for whole transcriptome sequencing (whole RNA-seq), the method for producing a non-ribosomal RNA-containing sample of the present invention can be carried out to obtain a non-ribosomal RNA-containing sample selectively containing mRNA. In a preferred embodiment, the method for producing a non-ribosomal RNA-containing sample is carried out in a sample containing mRNA and ribosomes (a sample in which a lysate is obtained in the presence of cycloheximide, and the RNA is fragmented after polyA selection of the mRNA), comprising a step (a) of peeling off ribosomal subunits and mRNA, and a step (b) of removing the ribosomal subunits peeled off in step (a) to obtain a non-ribosomal RNA-containing sample. This allows the production of a non-ribosomal RNA-containing sample containing fragments of mRNA that were expressed at the time of preparing the lysate to be obtained. A sequencing library for total RNA-seq can be prepared by subjecting a non-ribosomal RNA-containing sample to denaturing polyacrylamide gel electrophoresis together with an RNA size marker, excising RNA of a length appropriate for the sequencing platform, for example, RNA of 26 nt to 500 nt in length, purifying it from the gel, and then providing a linker, reverse transcribing it into cDNA, circularizing it, amplifying it by PCR, and adding a barcode according to the examples described below. The addition of a linker, adapter, or barcode, reverse transcription, and PCR amplification reaction after excising the RNA of the desired length and purifying it from the gel can be performed according to any known method, preferably any known method used for analysis with a next-generation sequencer.

[0054] In one embodiment, the method for producing a non-ribosomal RNA-containing sample of the present invention is carried out to prepare a sequencing library for analysis of small RNAs such as tRNA, snRNA, and snoRNA, and for analysis of microRNA, to obtain a non-ribosomal RNA-containing sample containing tRNA, snRNA, snoRNA, and microRNA. In a preferred embodiment, the method for producing a non-ribosomal RNA-containing sample, which includes a step (a) of peeling off ribosomal subunits and mRNA in a sample containing mRNA and ribosomes, and a step (b) of removing the ribosomal subunits peeled off in step (a), is carried out to obtain a non-ribosomal RNA-containing sample. For example, a sequencing library for microRNA analysis can be prepared by subjecting a non-ribosomal RNA-containing sample to denaturing polyacrylamide gel electrophoresis together with an RNA size marker, cutting out RNAs of 18 nt to 30 nt in length and purifying them from the gel, and adding a linker to the non-ribosomal RNA, reverse transcribing them into cDNA, circularizing them, amplifying them by PCR, and adding a barcode, in accordance with the examples described below. After excising RNA of the desired length and purifying it from the gel, the addition of a linker, adapter, or barcode, reverse transcription, and PCR amplification reaction can be carried out according to any known method, preferably any known method used for analysis with a next-generation sequencer.

[0055] As a sequencing technique for performing sequence determination, a method using a next-generation sequencer can be adopted. The type of next-generation sequencer is not particularly limited, but examples include HiSeq2000 (Illumina), Genome Analyzer IIx (Illumina), and Genome Sequencer-FLX (Roche). RNA sequencing using a next-generation sequencer includes a step of immobilizing nucleic acid on a flow cell or on a microarray. In the process of sequencing, bridge amplification (particularly bridge PCR) can occur in the flow cell on which the nucleic acid is immobilized, or in the microarray in which the nucleic acid is immobilized.

[0056] RNA sequencing is accomplished using "sequencing by synthesis (SBS)" techniques, where SBS refers to a technique in which a nucleic acid of interest is sequenced by synthesizing a complementary strand of the nucleic acid. The SBS technique may be selected from the group consisting of "pyrosequencing," "sequencing by ligation," and "sequencing by extension." "Pyrosequencing" refers to a method of sequencing by detecting pyrophosphate produced upon incorporation of a nucleotide. "Sequencing by ligation" refers to a method of determining a nucleic acid sequence using ligase to identify the nucleotide present at a specified position in the nucleic acid sequence. "Sequencing by extension" refers to a nucleic acid sequencing method in which a primer is extended with a known or detectable nucleotide.

[0057] "Deep sequencing" can be employed as a sequencing technique for performing sequence determination. "Deep sequencing" refers to a method of sequencing multiple nucleic acids in parallel (Bentley et al., Nature 2008, 456: 53-59). In a typical sequencing protocol using "deep sequencing", nucleic acids (e.g., DNA fragments) are attached to the surface of a reaction platform (e.g., flow cell, microarray, etc.). The attached nucleic acids are amplified in situ and can be used as templates for synthetic sequencing (e.g., SBS) using detectable labels (e.g., fluorescent reversible terminator deoxyribonucleotides). Representative reversible terminator deoxyribonucleotides can include 3'-O-azidomethyl-2'-deoxynucleoside triphosphates of adenine, cytosine, guanine, and thymine, each of which may be further labeled with a recognizable and removable fluorophore via a linker. Sequencing can be performed by either single-read or paired-end methods.

[0058] In the process of determining the base sequence of RNA in a non-ribosomal RNA-containing sample, when the base sequences of various sequencing libraries are determined, the sequence reads are aligned to the reference sequence, and after alignment, various analyses can be performed, such as identification of single nucleotide polymorphisms (SNPs) and insertions and deletions (indels), counting the number of reads for RNA methods, phylogenetic evolutionary analysis, and metagenomic analysis.

[0059] In the process of determining the base sequence of RNA in a non-ribosomal RNA-containing sample, the presence of sequence reads from potentially contaminating rRNA is also revealed by aligning them to a reference sequence.

[0060] <Kit> The kit used to carry out the method for producing a non-ribosomal RNA-containing sample of the present invention includes a reagent for peeling off ribosomal subunits and mRNA, and a means for removing ribosomal subunits. The reagent for peeling off ribosomal subunits and mRNA may be a chelating agent, and examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), glycoletherdiaminetetraacetic acid (EGTA, GEDTA), etc., and ethylenediaminetetraacetic acid (EDTA) is particularly preferred. The means for removing ribosomal subunits may be a centrifugal ultrafiltration filter unit composed of a tube equipped with an ultrafiltration membrane. The centrifugal ultrafiltration filter unit may be a double-structured centrifugal ultrafiltration tube further inserted into the tube. EXAMPLES

[0061] The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples. In this specification, unless otherwise specified, "%" and the like are based on mass, and numerical ranges are described as including their endpoints. The materials and equipment used in the examples are described at the end of the examples.

[0062] Example 1 Preparation of sequencing libraries by standard methods

[0063] A ribosome profiling library was prepared from HEK293 cells (ATCC, cat. no. CRL-1573) according to the procedure described in McGlincy NJ et al. 2017 (Non-Patent Document 3). The protocol based on McGlincy NJ et al. 2017 (Non-Patent Document 3) is described below. In this specification, the protocol described in Example 1 is referred to as the "standard method."

[0064] 1. Preparation of Lysate <Cycloheximide treatment> [Table 1] Prepare the above buffer and keep it chilled. Add the following reagents immediately before use:

[0065] [Table 2]

[0066] 1. Rinse the cells in a 10 cm dish with 5 mL of cold PBS. 2. Thoroughly discard the PBS and add 400 μL of lysis buffer (EDTA-free) and spread it evenly. 3. Dissociate the cells by pipetting and transfer them to a DNA Lobind tube. Wash with 200 μL of Lysis buffer. 4. Add 7.5 μL of 2 U / μL Turbo DNase I and place on ice for 10 minutes. 5. Centrifuge at 20,000 g for 10 min at 4℃. 6. Transfer the supernatant to a tube and mix by inversion. 7. Dispense one 5 μL vial for concentration measurement and the remaining 100 μL aliquots, flash freeze in liquid nitrogen, and store at -80℃.

[0067] <RNA Concentration Measurement Qubit RNA BR Assay Kit> 1. Prepare 200 μL of Working Solution × (2 for standards + 4 for samples). Working Solution = Qubit RNA BR Reagent 1 μL : Qubit RNA BR Buffer 200 μL 2. Dispense 190 μL for each standard and 199 μL for each sample into 0.5 mL tubes. Add 10 μL of each standard reagent #1 and #2, and 1 μL of each sample, vortex, and spin down. Let stand at room temperature for 2 min. 3. Measure using a Qubit 2.0 Fluorometer and select RNA BR Assay. Measure standards and samples. *It will reach 200 - 300 ng / μL from one 10 cm dish of 80% confluent HEK cells. (For 600 μL of lysate)

[0068] 2. RNase Digestion ~ Ultracentrifugation (Sucrose Cushion) · Prepare a heat block at 25°C · Cool the rotor of the ultracentrifuge

Table 3

[0069] Once the sucrose has dissolved, cool it on ice and add the following just before use.

Table 4

[0070]

Table 5

[0071] 1. Take RNA in a tube and make it up to 298 μL with Lysis buffer. 2. Add 2 μL of RNase I each time, mix gently, and incubate at 25 °C for 45 min in a heat block (perform precisely to avoid differences in reaction time between samples). 3. Transfer to ice and immediately add 10 μL (200 U) of SUPERase In (RNase inhibitor) to each tube. (Transferring to ice first can stop the reaction as cooling has this effect.) 4. Transfer 300 μL of the RNase I-digested sample to an ultracentrifuge tube. 5. Slowly pour 900 μL of Sucrose cushion buffer from below the sample to form two layers. 6. Centrifuge at 100,000 rpm at 4 °C for 1 h using a TLA110 rotor to obtain a ribosome pellet. 7. Discard the supernatant from the upper part of the liquid surface without disturbing the pellet.

[0072] 3. Footprint Fragment Purification <Direct RNA Recovery> Add 300 μL of TRIzol reagent to the pellet (until the pellet is visible), dissolve well (referred to as TRIzol sample), and transfer to a DNA Lobind tube.

[0073] <Direct-zol MicroPrep kit Column Purification>

Table 6

[0074] 1. Transfer to the column and centrifuge at 12,000 g for 1 min at 4 °C. The receiving tube should be discarded after each use. 2. Add 400 μL of PreWash buffer and centrifuge at 12,000 g for 1 min at 4 °C. Repeat this twice. 3. Add 700 μL of Wash Buffer and centrifuge at 12,000 g for 1 min at 4°C. 4. Perform a blank centrifugation at 12,000 g for 5 min at 4°C. 5. Add 6 μL of RNase-free water and centrifuge at 12,000 g for 2 min at 4°C. 6. Add 6 μL of 2x RNA Loading Buffer.

[0075] <RNA Size Marker Preparation and Electrophoresis>

Table 7

[0076] 1. Treat the RNA size marker and samples in a heat block at 95°C for 3 min → on ice for 2 min. For the RNA size marker, the Upper size marker oligoribonucleotide NI800 (34 nt) and Lower size marker oligoribonucleotide NI801 (26 nt) described in Non-Patent Document 3 (McGlincy and Ingolia, 2017, Methods, 126, 112 - 129) were used. 2. Use a WAKO SuperSep RNA 15% gel.

[0077] 3. After cleaning the wells, apply the samples. Electrophorese at a constant 10 mA for 50 min. 4. Stain the gel with 50 mL of 1 x TBE + 5 μL of SYBRGold (10,000-fold dilution). Shake on a shaker for 3 min. 5. Place on a blue light and check the bands. Cut out the area between 26 bp - 34 bp of the sample. 6. Also cut out the marker.

[0078] <RNA Extraction from Gel>

Table 8

[0079] 1. Crush the gel pieces in a 1.5 mL tube with a pestle. 2. Add 400 μL of RNA gel extraction buffer and wash off the gel on the pestle. 3. Freeze at -80℃ for 30 min or in liquid nitrogen. 4. Mix by inversion at room temperature for at least 2 hours. 5. Place the Spin-X column into a DNA Lobind 1.5 mL tube and transfer the gel solution using a wide tip. 6. 10000g, 1min, 4℃ 7. Add 3 μL of GlycoBlue and 500 μL of isopropanol and mix well. 8. Place in the freezer for 1 h, then centrifuge at 20,000 g, 30 min, 4°C. 9. Discard the supernatant and rinse with 70% ethanol. 10. Add 7 μL of 10 mM Tris pH 7.5 to the pellet and dissolve.

[0080] 4. Preparation of 20 μM preadenylated linker 1. Prepare the following solutions in 8 tubes: [Table 9] Treat at 65℃ for 1 hour, then at 85℃ for 5 minutes. The 5'p-linker-ddC primers used in the above table were NI-810 to NI-817 (Table 8) described in Non-Patent Document 3 (McGlincy and Ingolia, 2017, Methods, 126, 112-129).

[0081] 2. Oligo clean & Concentrator column purification [Table 10]

[0082] Transfer to a column and centrifuge at 12,000 g for 1 min. Add 750 μL of wash buffer and centrifuge at 12,000 g for 1 min. Centrifuge empty at 12,000 g for 5 min. Elution with 6 μL of RNase-free water. PAUSE POINT -20℃

[0083] 5. Dephosphorylation and Linker Ligation <Dephosphorylation> 1. Mixture preparation: Marker + 4 samples [Table 11] 2. Transfer the sample to an 8-tube strip and treat at 95℃ for 2 min, then on ice for 3 min. 3. Dispense 3 μL of the mixture into individual aliquots and place at 37℃ for 1 h. PAUSE POINT -80℃

[0084] <Linker ligation> -Attach a different linker to each sample. Make a note of it. Any linker can be attached to the marker 1. After dephosphorylation, treat at 95℃ for 2 min, then on ice for 3 min. [Table 12]

[0085] 2. Dispense 9 μL into each sample. 3. Add 1 μL of preadenylated linker (20 μM) and mix. 4. 22℃ 3h → 4℃ processing. 5. Purify using Oligo clean column (purify as described above). 6. Elution 6 μL + 2 x RNA Loading Buffer 6 μL PAUSE POINT -80℃

[0086] <Preparation for electrophoresis> (1) Linker only [Table 13] (2) Marker after linker ligation (3) Sample after linker ligation 1. Heat block at 95℃ for 3 min, then place on ice for 2 min. 2. Run as described above. 3. Excise the sample and marker after linker ligation. *Since the excised samples each have a different linker, they can be mixed together afterwards. 4. RNA extraction from gel (as described above)

[0087] 6. Reverse transcription reaction The RT primer used was the reverse transcription primer NI-802 described in Non-Patent Document 3 (McGlincy and Ingolia, 2017, Methods, 126, 112-129). 1. Prepare the following in an 8-tube array: (1) RT primer only (RNase-free water 10 μL) or (2) RT primer + linker (RNase-free water 9.5 μL + 20 μM liker 0.5 μL) or (3) Marker (after linker ligation) 10 μL or (4) Sample (after linker ligation) 10 μL + 1.25 uM RT primer NI802 2 μL Total 12 μL 6℃ 5 min → on ice 5 min

[0088] 2. Dispense 8 μL of the mixture into each of (1)~(4) + RT primer [Table 14]

[0089] 3. Treat at 50℃ for 30 minutes. 4. Add 2.2 μL of 1 M NaOH each time, mix, and heat at 70℃ for 20 min. 5. Oligo clean & Concentrator column purification

[0090] Add 28 μL of RNase-free water to make 50 μL. Purify as described above. Elution 6μL + 2x RNA Loading Buffer 6μL PAUSE POINT -20℃

[0091] <Preparation for electrophoresis> · Because the size of the annealed linker and RT primer overlaps with the sample size, heat in a heat block at 100℃ for 5 min. → Place on ice before electrophoresis. DNA gel extraction buffer (prepare during electrophoresis) [Table 15]

[0092] Add DNA gel extraction buffer to the excised gel to extract DNA, perform isopropyl precipitation as described above, and add 12 μL of 10 mM Tris pH 7.5 to dissolve the pellet.

[0093] 7. Circularization 1. Dispense 8 μL of the mixture into 8 tubes. [Table 16] 2. Dispense 12 μL of sample into each well. 3. Treat at 60℃ for 1 hour, then at 80℃ for 10 minutes. PAUSE POINT -20℃

[0094] 8. PCR Amplification and Barcode Addition The samples were subjected to varying cycles (6, 8, and 10 cycles) and bands with fewer non-specific bands were excised. Control (1) RT primer only (2) RT primer + linker (3) Marker (after linker ligation) requires only 8 cycles. 1. Prepare 100 μL of sample reaction solution and divide 33 μL into 3 wells (6, 8, 10 cycles)

[0095] As a control, 100 μL of reaction mixture without template was prepared and divided into three wells, and 1.7 μL of template was added to each well (8 cycles). If libraries are to be pooled after PCR, a different Rv primer (NI822-826, described in Non-Patent Document 3 (McGlincy and Ingolia, 2017, Methods, 126, 112-129)) is used for each library as necessary. (Number of samples that can be pooled = linker barcode x PCR primer barcode) [Table 17] The NI798 Fw primer and NI799 Rv primer were the forward library PCR primer NI-798 described in Non-Patent Document 3 (McGlincy and Ingolia, 2017, Methods, 126, 112-129) and the indexed reverse library PCR primer NI-799 in Table 9.

[0096] 2. After starting PCR, collect the tubes at the end of each extension cycle. 1) Process at 98℃ for 30 seconds. 2) [98℃ 10 s, 65℃ 10 s, 72℃ 5 s] x6, x8, x10 (collect 2 cycles each) 3) Treat at 72℃ for 5 minutes. 4) Place at 4℃. PAUSE POINT -20℃

[0097] 9. Gel purification of PCR products Do not denaturate the PCR product. Use SuperSep DNA, 15% gel. Use 6x non-denaturing purple loading dye. <Preparation for electrophoresis>

[0098] Mix 33 μL of sample + 6.5 μL of 6 x non-denaturing purple loading dye, and run 19 μL each in 2 wells. Mix 33 μL of RT primer, linker, and marker + 6.5 μL of 6 x non-denaturing purple loading dye, and run 10 μL of each in 2 wells. Run at 20 mA for 1 h for 20 min and stain the gel as described above. Check the optimal cycle number and excise the bands

[0099] <DNA Extraction from Gel> 1. Crush the gel pieces in 2 wells with a pestle and add 230 μL of DNA gel extraction buffer. 2. Freeze at -80 °C or in liquid nitrogen, then invert and mix for over 2 h at room temperature. Remove the gel pieces with a Spin-X column. 3. Purify with a NucleoSpin Gel and Clean column

Table 18

[0100] 4. Transfer to the column and centrifuge at 11,000 g for 1 min at 4 °C. 5. Wash with 700 μL of Buffer NT3 and centrifuge at 11,000 g for 1 min at 4 °C. Repeat 2 times. 6. Perform an empty centrifugation at 11,000 g for 5 min. 7. Add 17 μL of NE buffer and let stand at room temperature for 1 min. 8. Centrifuge for elution at 11,000 g for 1 min at 4 °C.

[0101] 10. Quality Check by MultiNA

[0102] <Measure in ultra-high sensitivity mode> · 1 / 25 Gel Star (diluted 25-fold with TE) · 1 / 5 Shimadzu Marker DNA-1000 (diluted 5-fold with H2O) · 1 / 50 100 bp DNA Ladder (diluted 50-fold with TE) 1. Preparation of separation buffer

Table 19

Table 20

[0103] Example 2 Preparation of sequencing libraries by ribosome splitting method In this example, a ribosome profiling library was prepared by stripping ribosomal subunits and removing ribosomes by ultrafiltration.

[0104] As in the method described in Example 1, a lysate is prepared from cells (section 1. Preparation of lysate in Example 1), which is digested with RNase and then subjected to ultracentrifugation to obtain a pellet of ribosomes (section 2. RNase digestion to ultracentrifugation (sucrose cushion) in Example 1). In this Example 2, instead of the <direct RNA recovery> described in section 3. Footprint Fragment purification in Example 1, the following <detachment of ribosomes and ultrafiltration> is performed.

[0105] <Ribosome Detachment and Ultrafiltration> 1. Prepare a pellet suspension according to the table below and place on ice for approximately 5 minutes. [Table 21]

[0106] 2. Place the ribosome pellet in an ultrafiltration tube and resuspend in 150 μL pellet suspension. The EDTA in the pellet suspension separates the ribosomes into large subunits, small subunits, and footprints.

[0107] 3. Transfer the mixture to a filter cup of AMICON® ULTRA 0.5 mL - 100 KDa cutoff (Millipore, cat. no. UFC510024) equipped with an attached reservoir tube, and centrifuge at 14,000 g for 10 minutes at 4°C.

[0108] 4. Discard the upper filter cup, and mix 360 μL of TRIzol LS reagent with the flow-through of the reservoir tube (referred to as TRIzol sample).

[0109] 5. Perform <Direct-zol MicroPrep kit column purification> in the section of "3. Footprint Fragment Purification" in Example 1. Subsequently, conduct the experiment as described in the sections from "4. Preparation of 20 μM Preadenylated linker" to "10. Quality Check by MultiNA" in Example 1.

[0110] (Example 3) Preparation of Sequencing Library by "Standard Method + rRNA Depletion" In this example, an rRNA Depletion step was added to the "Standard Method" described in Example 1 for sample preparation.

[0111] Conduct the experiment as described in the sections from "1. Preparation of Lysate" to "5. Dephosphorylation and Linker Ligation" in Example 1. Before the "6. Reverse Transcription Reaction" in Example 1, in "5. Dephosphorylation and Linker Ligation", rRNA Depletion was performed on the linker-ligated RNA sample as follows.

[0112] <Ribosomal RNA Depletion> (Ribo-Zero treatment) For Ribo-Zero treatment, four samples were treated together and dissolved in 10 mM Tris pH 7.5 to a total volume of 26 μL.

[0113] 1. Transfer the beads to a 225 μL tube and place on a magnet. 2. Discard the supernatant and wash twice with 225 μL of RNase-free water. 3. Suspend in 60 μL of Resuspension solution. Keep at room temperature. 4. Prepare the following solution in a 1.5 mL tube. [Table 22] Treat at 68℃ for 10 min, then at room temperature for 5 min.

[0114] 5. Add 65 μL of the prepared beads, pipette, and vortex for 10 s. Leave at room temperature for 5 min, then vortex for 10 s and place on a magnet. 6. Transfer the supernatant to a new tube. 7. Purify using Oligo clean & Concentrator column.

[0115] [Table 23]

[0116] (The column can only hold up to 800 μL, so pass the solution through the column in two separate runs.) Purify as described above and elute with 10 μL of RNase-free water.

[0117] Example 4 Preparation of sequencing libraries by "Ribosome splitting method + rRNA depletion" In this example, samples were prepared by adding an rRNA Depletion step to the "Ribosome splitting method" described in Example 2.

[0118] The experiment is carried out as described in "1. Preparation of lysate" to "5. Dephosphorylation and Linker Ligation" of Example 1, except that instead of the "direct RNA recovery" described in "3. Footprint Fragment Purification" of Example 1, "ribosome detachment and ultrafiltration" is carried out. Prior to "6. Reverse transcription reaction" of Example 1, in "5. Dephosphorylation and Linker Ligation", rRNA Depletion is carried out for the RNA sample that has been subjected to linker ligation as described in Example 3.<Ribosomal RNA Depletion> This was performed as described in (Ribo-Zero treatment).

[0119] Example 5 The libraries prepared and quality checked in Examples 1 to 4 were subjected to deep sequencing using HiSeq4000 (Illumina). The results of the number of reads for the sequencing libraries prepared by the standard method, the ribosome splitting method, the standard method + rRNA depletion, and the ribosome splitting method + rRNA depletion are shown in Figure 2. "Mapped" in Figure 2 means the number of reads mapped to the protein coding region (CDS), which corresponds to the number of ribosomes in the mRNA.

[0120] Ribosome profiling of a library prepared by the standard method from HEK293 cells yielded 9.2 × 10 rRNA reads. 5 The reads per million (RPM) for 92% of the library were useful, with only 5.4% (0.54 × 10 5 RPM) (Figure 2, Standard method).

[0121] rRNA-subtraction oligonucleotides that can hybridize to rRNA and be trapped on magnetic beads have been used to deplete rRNA reads (Ingolia et al., 2009, Science, 324, 218-23; Weinberg et al., 2016, Cell Rep, 14, 1787-1799; McGlincy and Ingolia, 2017, Methods, 126, 112-129). This rRNA depletion strategy using rRNA-subtraction oligonucleotides reduced rRNA contamination and reduced the number of rRNA reads by 7.7 × 10 5 The RPM library yielded 77% and mRNA reads were 1.8 × 10 5 The RPM library increased by 18% (Figure 2, Standard method + rRNA depletion).

[0122] On the other hand, the number of mRNA reads obtained by the ribosome splitting method was 2.3 × 10 5 In the RPM and library, the number of reads increased by 23% (Figure 2, Ribosome splitting method). Furthermore, when the Ribosome splitting method was combined with rRNA depletion, the number of reads of mRNA increased by 5.0 × 10 5 RPM increased by 50% in the library (Figure 2, Ribosome splitting method + rRNA depletion).

[0123] The standard method, ribosome splitting method, standard method + rRNA depletion, and ribosome splitting method + rRNA depletion were each repeated twice separately to prepare libraries and perform ribosome profiling. The Pearson correlation coefficient was calculated for each method and for the two repetitions for the number of mRNA reads. Pearson's correlation coefficient is a dimensionless quantity and takes values ​​between -1 and 1. A value between 0.7 and 1 is considered to be a strong correlation. The correlation coefficients are shown in Figure 3. The number of mRNA reads was reproducible when repeated twice with each method, and a high correlation was observed between the data of the four methods mentioned above.

[0124] material HEK293 cells (ATCC, cat. no. CRL-1573) DMEM (1x) + GlutaMAX-I (ThermoFisher Scientific, cat. no. 10566-016), with 10% FBS before use. 0.05% Trypsin-EDTA (ThermoFisher Scientific, cat. no. 25300-54) Cycloheximide 100 mg / ml (Sigma / Aldrich, cat. no. C4859-1ML) D-PBS (-)(1x) (nacalai tesque, cat. no. 14249-24) RNase-free water, molecular biology grade (Millipore, cat. no. H20MB1001) or (ThermoFisher Scientific cat. no. 10977-015) 1 M Tris-HCl pH 7.5, molecular biology grade (Wako Pure Chemical Industries, Ltd., cat. no. 318-90225) 5 M NaCl, molecular biology grade (nacalai tesque, cat. no. 06900-14) 1 M MgCl2, molecular biology grade (nacalai tesque, cat. no. 20942-34) Turbo DNase, 2 U / μl (ThermoFisher Scientific, cat. no. AM2238) Triton X-100, molecular biology grade (nacalai tesque, cat. no. 12967-32) Qubit RNA BR Assay kit (ThermoFisher Scientific, cat. no. Q10210) SUPERase·In, 20 U / μl (ThermoFisher Scientific, cat. no. AM2694) Sucrose, molecular biology grade (Wako Pure Chemical Industries, Ltd., cat. no. 198-13525) 3 M NaOAc pH 5.2, molecular biology grade (nacalai tesque, cat. no. 06893-24) RNase I, 10 U / μl (Epicentre, cat. no. N6901K) 13 x 56 mm polycarbonate ultracentrifuge tube (Beckman Coulter, cat. no. 362305) Direct-zol RNA MicroPrep (Zymo Research, cat. no. R2062) TRIzol (ThermoFisher Scientific, cat. no. 15596018) or other Direct-zol compatible reagent Ethanol, molecular biology grade (Wako Pure Chemical Industries, Ltd., cat. no. 054-07225) Isopropanol, molecular biology grade (Wako Pure Chemical Industries, Ltd., cat. no. 168-21675) GlycoBlue, 15 mg / ml (ThermoFisher Scientific, cat. no. AM9515) 0.5 M EDTA, molecular biology grade (Wako Pure Chemical Industries, Ltd., cat. no. 311-90075) 2× RNA Loading Buffer without Ethidium Bromide (Wako Pure Chemical Industries, Ltd. cat. no. 182-02571) SuperSepRNA, 15%, 17well (Wako Pure Chemical Industries, Ltd. cat. no. 194-15881) 10,000x SYBR Gold (ThermoFisher Scientific, cat. no. S11494) UltraPure 10% SDS (ThermoFisher Scientific, cat. no. 15553-027) T4 polynucleotide kinase (New England Biolabs, cat. no. M0201S). Supplied with 10x T4 polynucleotide kinase buffer. T4 RNA Ligase 2, truncated K227Q (New England Biolabs, cat. no. M0351S). Supplied with PEG 8000 50% w / v and 10x T4 RNA ligase buffer Preadenylated linkers at 20 μM Oligo Clean & Concentrator (Zymo Research, cat. no. D4060) 10 mM dNTP mix (New England Biolabs, cat. no. N0447L) ProtoScript II (New England Biolabs, cat. no. M0368L). Supplied with 5x first-strand buffer and 0.1 M DTT 1M Sodium hydroxide (nacalai tesque, cat. no. 37421-05) CircLigaseII ssDNA ligase (Epicentre, cat. no. CL9025K). Supplied with 10x CircLigaseII buffer, 5 M Betaine, and 50 mM MnCl2. Phusion polymerase (New England Biolabs, cat. no. M0530S). Supplied with 5x HF buffer. Gel Loading Dye, Purple (6×) (New England Biolabs, cat. no. B7024S) SuperSep DNA, 15%, 17 well (Wako Pure Chemical Industries, Ltd., 190-15481) DNA-1000 kit (SHIMAZU BIOTECH) GelStar Nucleic Acid Gel Stain 10,000× (LONZA, cat. no. 50535) 100 bp DNA Ladder (TAKARA BIO, cat. no. 3407A) NucleoSpin Gel and PCR Clean-up (TAKARA, cat. no 740609.250)

[0125] Equipment DNA LoBind Tube 1.5 mL (eppendorf, cat. no. 022431021) 8-strip PCR tube with lid (BIO-BIK, cat. no. 3247-00) Nunc 50mL Conical Sterile Polypropylene Centrifuge Tubes (ThermoFisher Scientific, cat. no. 339652) Eppendorf Tubes 5.0 mL (eppendorf, cat. no. 0030122313) Low retention filter tips (greiner bio-one, cat. nos. 771265, 773265, 738265, and 750265) Short 10 μl filter tips (watson, cat. no. 1252-207CS) Wide Bore 200 μl filter tips (Axygen, cat. no. TF-205-WB-R-S) Gel loading 20 μl filter tips (ThermoFisher Scientific, cat. no. 2155P) Refrigerated microcentrifuge (TOMY, cat. no. MX-307) Qubit 2.0 Fluorometer (ThermoFisher Scientific) Optima MAX-TL Ultracentrifuge (Beckman, cat. no. A95761) TLA 110 rotor (Beckman, cat. no. 366735) Dry block heater (Major science, cat. no. MC-0203) EasySeparator (Wako Pure Chemical Industries, Ltd. cat. no. 058-07681) Electrophoresis power supply (Amercham Biosciences, cat. no. EPS301) Blue light illuminator and orage filter cover (NA). A standard UV transilluminator can be used instead. Razors (Feather, cat. no. FAS-10) or (Feather, cat. no. No 11 stainless steel) Spin-X centrifuge tube filter 0.22 μM (costar, cat. no. 8160) Thermal cycler (Applied Biosystems, cat. no. 2720) DynaMag-2 separation rack (ThermoFisher Scientific, cat. no. 12321D) MixMate (eppendorf) MultiNA (SHIMAZU BIOTECH) Disposable homogenizer pestle R-1.5 (ASONE, cat. no. 1-2955-01)< / super>

Claims

1. In a sample containing mRNA and ribosomes, Mg 2+ (a) separating the ribosomal subunits and the mRNA using a chelating agent; and The method includes a step (b) of removing the ribosome subunits detached in the step (a) by ultrafiltration or size exclusion chromatography; Method for producing non-ribosomal RNA-containing samples.

2. 2. The method for producing a non-ribosomal RNA-containing sample according to claim 1, further comprising a step of degrading RNA or a step of fragmenting RNA in a sample containing mRNA and ribosomes.

3. The Mg 2+ 3. The method according to claim 1 or 2, wherein the chelating agent is at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA) and glycol ether diaminetetraacetic acid (EGTA, GEDTA).

4. A step of obtaining a non-ribosomal RNA-containing sample by carrying out the method for producing a non-ribosomal RNA-containing sample according to any one of claims 1 to 3; and Determining the base sequence of RNA in a sample containing non-ribosomal RNA A method for analyzing non-ribosomal RNA, comprising:

5. Mg 2+ Chelating agents, and including an ultrafiltration membrane or a size exclusion chromatography column; A kit used for carrying out the method for producing a non-ribosomal RNA-containing sample according to any one of claims 1 to 3.

Images