CN116463398B - Specific probe set combined with chicken ribosomal RNA and application thereof - Google Patents
Specific probe set combined with chicken ribosomal RNA and application thereof Download PDFInfo
- Publication number
- CN116463398B CN116463398B CN202310713150.6A CN202310713150A CN116463398B CN 116463398 B CN116463398 B CN 116463398B CN 202310713150 A CN202310713150 A CN 202310713150A CN 116463398 B CN116463398 B CN 116463398B
- Authority
- CN
- China
- Prior art keywords
- seq
- rna
- chicken
- probe
- reaction
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108020004418 ribosomal RNA Proteins 0.000 title claims abstract description 60
- 239000000523 sample Substances 0.000 title claims abstract description 56
- 241000287828 Gallus gallus Species 0.000 title claims abstract description 32
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims abstract description 41
- 102100034343 Integrase Human genes 0.000 claims abstract description 15
- 108010008532 Deoxyribonuclease I Proteins 0.000 claims abstract description 12
- 102000007260 Deoxyribonuclease I Human genes 0.000 claims abstract description 12
- 238000001816 cooling Methods 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims description 29
- 239000011324 bead Substances 0.000 claims description 28
- 239000000872 buffer Substances 0.000 claims description 12
- 238000009396 hybridization Methods 0.000 claims description 7
- 101710203526 Integrase Proteins 0.000 claims description 6
- 239000002787 antisense oligonuctleotide Substances 0.000 claims description 6
- 102000016911 Deoxyribonucleases Human genes 0.000 claims description 2
- 108010053770 Deoxyribonucleases Proteins 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 32
- 238000012163 sequencing technique Methods 0.000 abstract description 28
- 108020004999 messenger RNA Proteins 0.000 abstract description 8
- 238000013461 design Methods 0.000 abstract description 6
- 230000000692 anti-sense effect Effects 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 abstract description 4
- 108020005198 Long Noncoding RNA Proteins 0.000 abstract description 3
- 239000002699 waste material Substances 0.000 abstract description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 20
- 235000013330 chicken meat Nutrition 0.000 description 19
- 239000000203 mixture Substances 0.000 description 18
- 238000002156 mixing Methods 0.000 description 14
- 239000000243 solution Substances 0.000 description 13
- 239000006228 supernatant Substances 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 239000000047 product Substances 0.000 description 10
- 238000007664 blowing Methods 0.000 description 9
- 239000012295 chemical reaction liquid Substances 0.000 description 9
- 239000011259 mixed solution Substances 0.000 description 8
- 239000007788 liquid Substances 0.000 description 7
- 238000001514 detection method Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 238000007363 ring formation reaction Methods 0.000 description 6
- 230000029087 digestion Effects 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 108010090804 Streptavidin Proteins 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000001086 cytosolic effect Effects 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 108091027963 non-coding RNA Proteins 0.000 description 4
- 102000042567 non-coding RNA Human genes 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 3
- 239000003298 DNA probe Substances 0.000 description 3
- 239000013614 RNA sample Substances 0.000 description 3
- 238000010804 cDNA synthesis Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 230000002438 mitochondrial effect Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000011541 reaction mixture Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 241000206602 Eukaryota Species 0.000 description 2
- 108091046869 Telomeric non-coding RNA Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000002480 mineral oil Substances 0.000 description 2
- 235000010446 mineral oil Nutrition 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000002096 quantum dot Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 108020004463 18S ribosomal RNA Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000003837 chick embryo Anatomy 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000012165 high-throughput sequencing Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000012070 whole genome sequencing analysis Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a specific probe set combined with chicken ribosomal RNA and application thereof, belonging to the technical field of biology. The invention designs a specific antisense probe by referring to the sequence of chicken ribosomal RNA, combines the probe with the ribosomal RNA in total RNA to form a hybrid chain by a gradient cooling method, and eliminates the ribosomal RNA, genome DNA and the probe by using RNase H enzyme and DNase I enzyme. The purified RNA product obtained by the method can be used for sequencing mRNA and part of lncRNA containing a poly A structure through a commercial library-building sequencing kit, so that the sequencing data amount and the waste of samples are reduced, and the application of a transcriptome sequencing technology in chicken species is enhanced.
Description
Technical Field
The invention relates to a specific probe set combined with chicken ribosomal RNA and application thereof, belonging to the technical field of biology.
Background
Transcriptome sequencing is a high-efficiency experimental method widely applied to biology and genetics except whole genome sequencing at present, and the gene expression condition of organisms can be known through the transcriptome sequencing, so that key genes affecting development, reproduction or diseases are discovered. With the continued development of sequencing technology, various non-coding RNAs, such as long non-coding RNAs (lncrnas), micro non-coding RNAs (mirnas), and circular non-coding RNAs (circrnas), were determined to play an important role in the development of organisms. At present, mRNA and the expression quantity of lncRNA containing a poly A structure can be detected in total RNA of eukaryotes simultaneously through mRNA library building means, but research shows that ribosomal RNA (rRNA) in eukaryotes is divided into cytoplasmic ribosomal RNA and mitochondrial ribosomal RNA, which account for about 80% of total RNA, and the two types of RNA are high in expression abundance and highly conserved, so that the existence of rRNA leads to most sequenced data as invalid information. Although transcriptome sequencing is increasingly cheaper, the method of obtaining complete and effective information by increasing the amount of sequencing data is wasteful, and thus the rRNA in total RNA needs to be removed in advance for mRNA sequencing.
At present, three methods for removing ribosomal RNA mainly exist, wherein the first method uses oligo-DT magnetic beads to enrich mRNA with a Poly A structure, thereby indirectly realizing the effect of removing rRNA; the second method is to design a probe with streptavidin marked and rRNA complementary, and remove rRNA through streptavidin magnetic beads after combination; the third method designs a large number of rRNA antisense probes to bind to rRNA to form rRNA-DNA double strand, degrading rRNA by RNase H enzyme.
The first method is the currently mainstream transcriptome sequencing method, and there are a variety of products of the beads kit on the market, such as VAHTS mRNA Capture Beads of Nuo-only company and Oligo dT-Coated Magnetic Beads of the next holy company, which can effectively remove rRNA, but can result in non-coding RNA without poly A structure in total RNA and poor quality mRNA loss. The second method is most stable, but it is complicated to operate and expensive to use streptavidin-labeled probes and magnetic beads, and is not suitable for processing a large number of samples. The third method designs an rRNA sequence antisense probe system, combines the probe and target RNA to form a target RNA-DNA probe hybrid chain through gradient cooling, digests the target RNA in the hybrid chain through RNase H enzyme, and finally eliminates genome DNA and DNA probe by DNase I enzyme. The method is convenient to operate, but because of limited rRNA sequence consistency among species, most commercial kits are designed to perform rRNA removal on probes of human or mice through a third method, and the applicant finds out through search that China patent application 202010056986.X discloses a method for removing ribosomal RNA in total RNA of animals, which can only remove similar regions of ribosomal RNA of all species after use, cannot remove species-specific ribosomal RNA regions and cannot only target the chicken species. At present, no method for efficiently removing the ribosomal RNA of the chicken is available on the market, and the scientific research of the chicken species is greatly limited.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art, and provides a specific probe set combined with chicken ribosomal RNA, and the specific probe set is used for effectively removing the ribosomal RNA in chicken total RNA.
The invention solves the technical problems through the following technical scheme: first, a specific probe set combined with chicken ribosomal RNA is provided, which consists of 145 antisense probes, and the sequences of the antisense probes are SEQ ID NO.1-SEQ ID NO.145.
The invention further solves the technical problems through the following technical proposal, and the application of the specific probe group combined with the chicken ribosomal RNA comprises the following steps:
firstly, designing a specific antisense probe aiming at cytoplasmic ribosomal RNA and mitochondrial ribosomal RNA sequences of chickens, extracting total RNA of chickens, and combining the probe with rRNA of the chickens to form a target RNA-DNA probe hybridization double chain by a gradient cooling method;
secondly, removing target RNA in the RNA-DNA double strand by adopting RNase H enzyme;
thirdly, removing the genome DNA and the probe by DNase I enzyme;
and fourthly, recovering the purified RNA by using magnetic beads.
The hybridization system in the first step of the method is hybridization Buffer 3 mu L, probe 1 mu L, total RNA 1 mu g, total reaction volume 15 mu L, reaction condition is 105 ℃ heat cover, 95 ℃ 2min, 95 ℃ to 22 ℃ 0.1 ℃/s,22 ℃ 5min and the reaction is finished to 4 ℃.
The reaction system of RNase H enzyme treatment in the second step is as follows: 3 mu L of RNase H Buffer, 2 mu L of RNase H, 15 mu L of the product of the previous step and 20 mu L of the total volume; the reaction conditions were 37℃for 30min.
The reaction system of DNase I enzyme treatment in the third step is as follows: DNase I Buffer 27.5 mu L, DNase I2.5 mu L, the product of the last step 20 mu L, total volume 50 mu L; the reaction conditions were 37℃for 30min.
The invention designs a specific antisense probe aiming at a chicken ribosomal RNA sequence, combines the designed probe with rRNA in chicken total RNA to form a hybrid chain by a gradient cooling method, removes rRNA, genome DNA and the probe in the hybrid chain by RNase H enzyme and DNase I enzyme, uses a Qsep100 full-automatic nucleic acid protein analysis system to carry out quality inspection, and finally detects the residual condition of the rRNA by a library-building sequencing method. The invention also adds a complementary probe of ribosomal RNA in mitochondria, and supplements the original cytoplasmic ribosomal RNA removal work.
The beneficial effects of the invention are as follows: the RNA product obtained by the method can be used for sequencing mRNA and part of lncRNA containing a poly A structure through a library-building kit, so that the waste of sequencing data and samples is reduced, the transcriptome sequencing cost of an RNA sample is greatly reduced, and the application of the transcriptome sequencing technology in chicken is enhanced. The problem that a method for specifically removing ribosomal RNA in total RNA of chickens is lacking in the market is solved, although the rRNA removing method is slightly higher in time than the other two methods and needs a plurality of magnetic bead purification processes, the partial RNA information loss caused by an oligo-DT magnetic bead method and the high cost requirement of a streptavidin labeling method are effectively avoided, and the application of a transcriptome sequencing technology to chickens is facilitated.
Drawings
FIG. 1 shows the Qsep100 instrument detection results of total RNA samples that have not been subjected to the probe treatment of the present invention.
FIG. 2 shows the Qsep100 instrument detection results of total RNA samples treated with the probe of the present invention.
FIG. 3 is a comparison of the residual rates of ribosomal RNA after treatment with the inventive probe A and the commercial probe B.
Description of the embodiments
The following examples facilitate a better understanding of the present invention, but are not limited thereto. The following methods are conventional methods, and the reagent consumables used in the following are all purchased from reagent companies unless otherwise specified. The following experiments used three different samples and the results are shown as average values.
Hybridization Buffer, RNase H Buffer, RNase H, DNase I Buffer, DNase I, hieff NGS are all from the company of Hieff NGS MaxUp Human rRNA Depletion Kit, RNA cleaner beads, hieff NGS Ultima Dual-mode RNA Library Prep Kit, inc. of Saint Biotechnology (Shanghai). The double-Barcode cyclization kit, MGISEQ-2000RS sequencing slide and cPAS Barcode primers are all from Shenzhen Hua Dazhi manufactured technology Co., ltd.
Examples
The present example removes ribosomal RNA from chicken total RNA as follows:
1. designing and preparing probes
The cytoplasmic ribosomal RNA and mitochondrial ribosomal RNA sequences of chickens are obtained from NCBI websites, 145 antisense probes are obtained through design, and the probes are conserved among various chicken varieties and have compatibility.
The 145 probe sequence information is as follows (5 '-3') orientation:
| SEQ ID NO.1 | CAGGCCCGACCCTGCTTAGCTTCCGAGATCAGACGAGATC |
| SEQ ID NO.2 | AAGCCTACAGCACCCGGTATTCCCAGGAGGTCTCCCATC |
| SEQ ID NO.3 | CACATTAATTCTCGCAGCTAGCTGCGTTCTTCATCGACGC |
| SEQ ID NO.4 | CGTAGCCCCGGGAGGAACCCGGGGCCGCAAGTGCGTTCG |
| SEQ ID NO.5 | CACTGGGATGCGGATACTTGCATGTATATGTCTAGCAAAA |
| SEQ ID NO.6 | TCTTGGGCTACTGCTGAGTGTGCCTGATACCTGCTCCTTT |
| SEQ ID NO.7 | ATTGCTTAAGGTTAATTACTGCTGAGTACCCGTGGGGGT |
| SEQ ID NO.8 | TGTATGACCGCGGTGGCTGGCACAAGATTTACCAACCCTG |
| SEQ ID NO.9 | ATCTTAGCTGGTGCAGATAACATGTGGCCACTCTTTACGC |
| SEQ ID NO.10 | GTTGAGGCTAAGATGGATTTGGGTTGGGTTTAGGTGGATC |
| SEQ ID NO.11 | ATTTAGGGCTAGGCATAGTGGGGTATCTAATCCCAGTTTG |
| SEQ ID NO.12 | AGTCCTTAGAGTTTTAAGCGTTTGTGCTCGTAGTTCTCAG |
| SEQ ID NO.13 | GTGGATTATCGATTATAGAACAGGCTCCTCTAGGTGGGT |
| SEQ ID NO.14 | TTCTTTCATTAGAGGTGGGCTGGCGACGGCGGTATGTAGG |
| SEQ ID NO.15 | CCATTTCTCCCACCCCATAGGCTATACCTTGACCTGTCTT |
| SEQ ID NO.16 | ACTGCTAAATCCTCCTTCTAAGGGCGGGTTTCACATCCTT |
| SEQ ID NO.17 | ACGTGCCTCAGAGCCGTCTTAAAGTGAGCTTAGGGGGTA |
| SEQ ID NO.18 | TGATGTTGATGGCTTGTGAAGAGGGTGACGGGCGGTATGT |
| SEQ ID NO.19 | GCACCTTCCGGTACACTTACCTTGTTACGACTTGCCTCGT |
| SEQ ID NO.20 | TTTTATGTTAAGGGTACGAGTTTGTCGGGCTAGAGGGGGG |
| SEQ ID NO.21 | GGGGTAGTCTTTTCTATCGCCTATACTGGGACAATTTAT |
| SEQ ID NO.22 | TTTGCTTATGGTTTTCATTGCTATTTCATCTTTCCCTTGC |
| SEQ ID NO.23 | AGCTCACTTTGCTTGGTTGTTCTTGCTAAATCATGATGCA |
| SEQ ID NO.24 | GAGACGGGTTCGCTCAAATTTTAGCTGCTCGCAAGTAGCT |
| SEQ ID NO.25 | GTAACCAGCTATCACCCAGCTCGGTAGGCTTTTCACCTCT |
| SEQ ID NO.26 | GTTCTCAAGGTTGACAAAGGTGGGTGTCTTTAGGGGGTGG |
| SEQ ID NO.27 | TATCCGCTGGAGGAGGTTGTGTTCTTTTTCGATGGAGCTG |
| SEQ ID NO.28 | TGAGGGAGCTTTGACGCACTCTTTTGTTGGTGGCTGCTTG |
| SEQ ID NO.29 | TTCTTCTATTGTCATAGGTTAACCTGCTTTGGTTGAGGGA |
| SEQ ID NO.30 | ACGCCGTAGGAGGATAGGTTCCAGATTACTCATTTTAGCA |
| SEQ ID NO.31 | CGTATTGCTTGTTAGTGTGGGGGTATAAGTTGAGTTCTGT |
| SEQ ID NO.32 | GCCGAGTTCCTTCTGTAGGTTTTAATCATCCTGTGGGCGC |
| SEQ ID NO.33 | GCTGAAGGCTATGTTTTTGGGAAACAGTCGGGTCTTTGGT |
| SEQ ID NO.34 | GGTTAGGATACCGCGGCCGTTGAACTTTGGGGGTCACTG |
| SEQ ID NO.35 | GTCTCAATTTACGGGACAATTGATTGCGCTACCTTCGCAC |
| SEQ ID NO.36 | ACCTACAGGAGACAGTTAAGACCTCGTTTAGCCATTCAT |
| SEQ ID NO.37 | AGTTCCACAGGGTCTTCTCGTCTTATGTTCACATTCTCGT |
| SEQ ID NO.38 | CGACCAGGGGTTTATGTGTGGGTGGACCCAGTGGGGCTG |
| SEQ ID NO.39 | AGTGAAGAGTTGTGGTCTGTGGGTTTGGAGGATTTTTTT |
| SEQ ID NO.40 | TGGGGTAGCTTGGTCCATTGCTCAATTATATTGGGTCTGG |
| SEQ ID NO.41 | ACATCGAGGTCGTAAACCTCCTTGTCGATATGGGCTCTT |
| SEQ ID NO.42 | GAACCCTTAATAGCGGTTGCACCATTAGGTTGTCCTGAT |
| SEQ ID NO.43 | AACCGACCTGGATTGCTCCGGTCTGAACTCAGATCACGT |
| SEQ ID NO.44 | TGTGCTCAGTGGTATTGACCCCACTTCTCCGGTCCTTTC |
| SEQ ID NO.45 | TAGGAGTTGGGTGTGGGGGAGGGGTTCTTGGCAGTTGAG |
| SEQ ID NO.46 | CTTTGAGACAAGCATATGCTACTGGCAGGATCAACCAGGT |
| SEQ ID NO.47 | ACTGATTTAATGAGCCATTCGCAGTTTCACTGTACCGCCC |
| SEQ ID NO.48 | ATTACCACAGTTATCCAAGTAACGGGAGGGGAGCGACCA |
| SEQ ID NO.49 | GGTTGGTTTTGGTCTGATAAATGCACGCGTCCCCGGAGGT |
| SEQ ID NO.50 | CGTCGCCGCCACGGGGGCGTGCGATCGGCTCGAGGTTATC |
| SEQ ID NO.51 | TTACCCGTGGTCACCATGGTAGGCACAGACAGTACCATCG |
| SEQ ID NO.52 | CTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTCTCCGGAA |
| SEQ ID NO.53 | TCCTGTATTGTTATTTTTCGTCACTACCTCCCCGGGTCGG |
| SEQ ID NO.54 | GCCCTCCAATGGATCCTCGTTAAAGGATTTAAAGTGGACT |
| SEQ ID NO.55 | ATATACGCTATTGGAGCTGGAATTACCGCGGCTGCTGGC |
| SEQ ID NO.56 | CTCGCCTCGCGGCGGACCGCCAGCTCGATCCCAAGATCCA |
| SEQ ID NO.57 | GGGCCCCGCGGGACACTCAGTTAAGAGCATCGAGGGGGCG |
| SEQ ID NO.58 | TATTCCTAGCTGGAGTATTCCGGCGGCCAGCCTGCTTTGA |
| SEQ ID NO.59 | CCCGGCCGTCCCTCTTAATCATGGCCCCGTTTCCGAAAAC |
| SEQ ID NO.60 | AATGCTTTCGCTTTAGTTCGTCTTGCGCCGGTCCAAGAA |
| SEQ ID NO.61 | GACGGTATCTGATCGTCTTCGAACCTCCGACTTTCGTTCT |
| SEQ ID NO.62 | GAGCTGCCCGGCGGGTCATGGGAATAACGCCGCCGGATCG |
| SEQ ID NO.63 | GTCAATTCCTTTAAGTTTCAGCTTTGCAACCATACTCCCC |
| SEQ ID NO.64 | CCGGGCCGGGTGAGGTTTCCCGTGTTGAGTCAAATTAAGC |
| SEQ ID NO.65 | ACGGCCATGCACCACCACCCACGGAATCGAGAAAGAGCT |
| SEQ ID NO.66 | ACTAGTTAGCATGCCAGAGTCTCGTTCGTTATCGGAATT |
| SEQ ID NO.67 | TCAATCTCGGGTGGCTGAACGCCACTTGTCCCTCTAAGAA |
| SEQ ID NO.68 | AGCCAGTCAGTGTAGCGCGCGTGCAGCCCCGGACATCTAA |
| SEQ ID NO.69 | CCCCGATCCCCATCACGAATGGGGTTCAACGGGTTACCCG |
| SEQ ID NO.70 | ACGCGAGCTTATGACCCGCACTTACTGGGAATTCCTCGT |
| SEQ ID NO.71 | ACCTCACTAAACCATCCAATCGGTAGTAGCGACGGGCGGT |
| SEQ ID NO.72 | TAGTCAAGTTCGACCGTCTTCTCGACGCTCCGGCAGGGCC |
| SEQ ID NO.73 | GATCCTTCCGCAGGTTCACCTACGGAAACCTTGTTACGA |
| SEQ ID NO.74 | TTCCTCCGCTGACTAATATGCTTAAATTCAGCGGGTCGCC |
| SEQ ID NO.75 | ATTCGGCGCTGGGCTCTTCCCTCTTCACTCGCCGTTACTG |
| SEQ ID NO.76 | CGAGAGCGGCGCCGGGGATGGGGGCTTCCGTACGCCACA |
| SEQ ID NO.77 | CGGCCTCACACCGTCCGCGGGCTGGGCCTCGATCAGAAG |
| SEQ ID NO.78 | GCTTTGGGCTGCATTCCCAAGCAACCCGACTCCGAGAAGC |
| SEQ ID NO.79 | CTTTCCCTTACGGTACTTGTTGGCTATCGGTCTCGTGCCG |
| SEQ ID NO.80 | GCGGACCCCACCCGTTTACCTCTTAACGGTTTCACGCCCT |
| SEQ ID NO.81 | CCGACCTTGGCCCGCCGGGTTGAATCCTCCGGGCCGACTG |
| SEQ ID NO.82 | CCGCCCCGGCGAAGGGGAGGGACGGAGGGGAGGCGGAGG |
| SEQ ID NO.83 | ATGCGCCCGACGGGGGCCGGACGCCGGCCGGGCGGCGGTC |
| SEQ ID NO.84 | CCGTCCCGGAGCCGGTCGCGGCGCACCGCCGCGGAGGAA |
| SEQ ID NO.85 | CCGGCGGCTATAACACCCGGCGGCCGCTCGCGCGGCGCCG |
| SEQ ID NO.86 | CGGTCCTCTCCCTCGGCCCCGGGATTCGGCGACGATCCG |
| SEQ ID NO.87 | CGACGCCGGTCCGGCCGCGGGGGGCCCTCCGGGGGGCCG |
| SEQ ID NO.88 | GCGGGGAACGGAGAGAGAGCGAGCGGCGGCGTACGGCGGC |
| SEQ ID NO.89 | CGGCGCCGCGCCGAGCCGCGCGCCGGACACCCGACCCCC |
| SEQ ID NO.90 | CGGCCTCGCCCGCGCCGGGCTGGAGGCGCGCGCCACACG |
| SEQ ID NO.91 | AACGGGCCGCCCCCGCGGGAACGGAGGCGGCCCGAACAGA |
| SEQ ID NO.92 | CGGGGCGCACTGAGCGCAGTCCGCCCCGGTCGGACAGCG |
| SEQ ID NO.93 | TAGCCGACGTCGCCGCGGACCCCGGGCGCCCTGGCGTGG |
| SEQ ID NO.94 | TTTCGCGCGAGCCGCCGACTCGCGCGCGTGCTAGACTCCT |
| SEQ ID NO.95 | CACCTCAGCCGGCGCGCGCCGGCCCTCACCTTCATTGCG |
| SEQ ID NO.96 | CGGCGAGGCGGGCGAGACGGGCCGGTGGTGCGCCCGGGG |
| SEQ ID NO.97 | CTGCCCAGGCATAGTTCACCATCTTTCGGGTCCTAGCAC |
| SEQ ID NO.98 | CCGGGTCGGACGACCGATTTGCACGTCAGGACCGCTACGG |
| SEQ ID NO.99 | GGAGGGAACCAGCTACTAGATGGTTCGATTAGTCTTTCGC |
| SEQ ID NO.100 | TAATCATTCGCTTTACCGGGTAAAACTGCACCGCCGCCCC |
| SEQ ID NO.101 | AGTTTGAGAATAGGTTGAGATCGTTTCGGCCCCAAGACCT |
| SEQ ID NO.102 | CCGGCTCCACGCCAGCGAGCCGGGCGTCTTACCCATTGAA |
| SEQ ID NO.103 | CGTTCGGTTCATCCCGCAGCGCCAGTTCTGCTTACCAAAA |
| SEQ ID NO.104 | CCTGCTGTCTAGATCAACCAACACCTTTTCTGGGCTCTGA |
| SEQ ID NO.105 | CAGGGCTAGTTGATTCGGCAGGTGAGTTGTTACACACTCC |
| SEQ ID NO.106 | CGACGGCCGGGTATGGGCCCGACGCTCCAGCGCCATCCAT |
| SEQ ID NO.107 | CAGGCGCACCGCAGCGGCCCTCCTACTCGTCGCGGCGTAG |
| SEQ ID NO.108 | TTGAATAGTTGCTACTACCACCAAGATCTGCACCTGCGGC |
| SEQ ID NO.109 | GACCGACTGACCCATGTTCAACTGCTGTTCACATGGAACC |
| SEQ ID NO.110 | CCTTTCGATCGGCTGAGGGCAACGGAGGCCATCGCCCGTC |
| SEQ ID NO.111 | CGGCGCCCGTCTCCGCCGCTCCGGATTCGGGGATCTGAA |
| SEQ ID NO.112 | AAAGAGAACTCTCCCCGGGGCTCCCGCCGGCTTCTCCGGG |
| SEQ ID NO.113 | TCGGGGCGAACCCGTTCCAGGGCGCCCGGCCCTTCACAAA |
| SEQ ID NO.114 | TTCACGGGCCAGCGAGAGCTCACCGGACGCCGCCGGAACC |
| SEQ ID NO.115 | GTTCACCTTGGAGACCTGCTGCGGATATGGGTACGGCCCG |
| SEQ ID NO.116 | CCTTATCCCGAAGTTACGGATCCGGCTTGCCGACTTCCCT |
| SEQ ID NO.117 | TTCGCGCCCCAGCCCGACCGACCCAGCCCTTAGAGCCAAT |
| SEQ ID NO.118 | GGAGCGGGGAAAGGGGGGGCGGGGGCGGGCGGCGCCTCG |
| SEQ ID NO.119 | CGCGCGTCCAGAGTCGCCGCCGCCGCGCCGCCCGCTGAC |
| SEQ ID NO.120 | CGAGCGGCGCCCGCCGCAGCTGGGGCGATCCACGGGAAG |
| SEQ ID NO.121 | CGGCCGGGACGACGGCCGACGGGAGGGGCGCCGGGAGCG |
| SEQ ID NO.122 | CGCCCCTCGCGCCCTCGCGAGGAGGGCGCTCGGGACGGG |
| SEQ ID NO.123 | CGCCACCGGCGGGCCCCCCCCCGCCGCCGCCGCGCCCGCG |
| SEQ ID NO.124 | TGCTAGGCGCCGGCCGAGGCGGGGCGCCGGCCCGGAGAC |
| SEQ ID NO.125 | GCTTTGTTTTAATTAAACAGTCGGATTCCCCTGGTCCGCA |
| SEQ ID NO.126 | TCAGAGCACTGGGCAGAAATCACATCGCGTCAACACCCGC |
| SEQ ID NO.127 | GCTACCTTAAGAGAGTCATAGTTACTCCCGCCGTTTACCC |
| SEQ ID NO.128 | AGTAGGTAGGGACAGTGGGAATCTCGTTCATCCATTCATG |
| SEQ ID NO.129 | AAGCTCAACAGGGTCTTCTTTCCCCGCTGATTCCGCCAAG |
| SEQ ID NO.130 | GTCGCGCGACCGCGGGGCCTCCCACTTATTCTACACCTCT |
| SEQ ID NO.131 | AAAACGATCAGAGTAGTGGTATTTCACCGGCGGCCGGGCC |
| SEQ ID NO.132 | CGCCGGGCGCTTGGCGCCAGAAGCGAGAGCCCCTCGGGG |
| SEQ ID NO.133 | TTGACAGGTGTACCGCCCCAGTCAAACTCCCCACCTGACG |
| SEQ ID NO.134 | TCTGGCCTCCCTGAGCTCGCCTTAGGACACCTGCGTTACG |
| SEQ ID NO.135 | AATCAAGATCAAGCGAGCTTTTGCCCTTCTGCTCCACGG |
| SEQ ID NO.136 | AAAAGTCAGAAGGATCGTGAGGCCCCGCTTTCACGGTCT |
| SEQ ID NO.137 | ATGAACGCTTGGCCGCCACAAGCCAGTTATCCCTGTGGTA |
| SEQ ID NO.138 | GCTTGGTGAATTCTGCTTCACAATGATAGGAAGAGCCGAC |
| SEQ ID NO.139 | ACTAACCTGTCTCACGACGGTCTAAACCCAGCTCACGTTC |
| SEQ ID NO.140 | GTCTGAACCTGCGGTTCCTCTCGTACTGAGCAGGATTACT |
| SEQ ID NO.141 | GCGTTCAGTCATAATCCCACAGATGGTAGCCTCGCTCCAG |
| SEQ ID NO.142 | TATCGCGAGCCCACCGAGGCGCCTCGGCGCTGCGGTATC |
| SEQ ID NO.143 | CGCTCCGGTCCCGACCACGAGCGGCGCTCCGCACCGACCG |
| SEQ ID NO.144 | ATGATTTAGCACCGGGTTCCCCACGAACATGCGGTACGCG |
| SEQ ID NO.145 | TCAATAGATCGCAGCGAGGGAGCTGCTCTGCTACGTACGA |
after all the probes are prepared, diluting and mixing to form a probe pool, and ensuring that the final concentration of each probe in the probe pool is 1 mu M. The total RNA of the sample was extracted from skin tissue of 13 embryo-age chick embryos using Trizol reagent, and the integrity of the total RNA was measured using Qsep100 apparatus, as shown in FIG. 1.
2. 1. Mu.g of total RNA was subjected to probe hybridization, and the system was prepared in the following ratio. Probes in the system are compared with commercial probes by using the designed probes of the invention respectively, and the rest conditions are kept consistent.
The solution was mixed and then immediately removed to the bottom of the tube and placed on a PCR instrument, and the reaction procedure was as follows:
3. digestion with RNase H enzyme was performed as follows:
the mixed solution was placed on a PCR apparatus at 37℃for 30min.
4. Digestion with DNase I enzyme
The mixed solution is placed on a PCR instrument after shaking and mixing evenly, and the temperature is 37 ℃ for 30min.
RNA purification
(5.1) balancing the RNA Cleaner magnetic beads for more than half an hour at room temperature, uniformly mixing by vortex vibration, preparing a fresh 80% ethanol solution by DEPC water, and standing at room temperature for later use.
(5.2) sucking 110. Mu.l of RNA Cleaner magnetic beads into the reaction liquid product, shaking and mixing uniformly, and incubating for 5min at room temperature.
(5.3) the PCR tube was placed on a magnetic rack, allowed to stand for 5min, and the supernatant was discarded after the solution was clarified.
(5.4) the PCR tube was placed on a magnetic rack all the time, rinsed with 80% ethanol solution, and after 30s, the supernatant was aspirated and discarded.
(5.5) repeating step (5.4) for two rinsing cycles.
(5.6) after the residual liquid was sucked up by a pipette, the beads were dried at room temperature for about 5 minutes.
(5.7) RNA elution: the PCR tube was removed from the magnetic rack, 6. Mu.L of DEPC water was added, the beads were thoroughly mixed, and left to stand at room temperature for 5min.
(5.8) the PCR tube was placed on a magnetic rack for adsorption for 5min, 4.25. Mu.L of the supernatant containing the purified RNA was placed in a new 200. Mu.L PCR tube, and the purified RNA was immediately subjected to library construction.
6. Construction of sequencing library
The present study utilizes Hieff NGS ® Ultima Dual-mode RNA Library Prep Kit.
(6.1) RNA fragmentation
Adding an equal volume of 2 XFrag/Prime Buffer into the supernatant in the step (5.8), fully blowing and uniformly mixing by using a pipettor, and then carrying out fragmentation according to the following procedure:
| temperature (temperature) | Time |
| 94℃ | 10 min |
(6.2) Synthesis of first strand cDNA
The reaction solution for first strand cDNA synthesis was prepared as follows:
| reagent name | Volume (mu L) |
| Reaction liquid in the last step | 8.5 |
| Strand Specificity Reagent | 3 |
| 1st Strand Enzyme mix | 1 |
| Total | 12.5 |
And (5) blowing and uniformly mixing by using a liquid transfer device, and instantaneously separating.
The PCR tube was placed on a PCR instrument and first strand cDNA synthesis was performed, the reaction procedure was as follows:
| temperature (temperature) | Time |
| Thermal cover 105 DEG C | On |
| 25℃ | 10 min |
| 42℃ | 15 min |
| 70℃ | 15 min |
| 4℃ | ∞ |
(6.3) Synthesis/end repair/addition A of second strand cDNA
The reaction solution for second strand cDNA synthesis was prepared as follows:
| reagent name | Volume (mu L) |
| Reaction liquid in the last step | 12.5 |
| 2nd Strand Buffer(dUTP) | 15 |
| 2nd Strand Enzyme Master Mix | 2.5 |
| Total | 30 |
And (5) blowing and uniformly mixing by using a liquid transfer device, and instantaneously separating.
The PCR tube was placed on a PCR instrument and the second strand cDNA was synthesized as follows:
| temperature (temperature) | Time |
| Thermal cover 105 DEG C | On |
| 16℃ | 30 min |
| 72℃ | 15 min |
| 4℃ | ∞ |
(6.4) connecting Joint
The reaction solution for preparing the connecting joint is as follows:
| reagent name | Volume (mu L) |
| Reaction liquid in the last step | 30 |
| Ligation Enhancer | 15 |
| Novel T4 DNA Ligase | 2.5 |
| DNA Adapter(5uM) | 2.5 |
| Total | 50 |
And (5) blowing and uniformly mixing by using a liquid transfer device, and instantaneously separating.
The PCR tube was placed on a PCR instrument and subjected to linker ligation, and the reaction procedure was as follows:
| temperature (temperature) | Time |
| Thermal cover | Off |
| 20℃ | 15 min |
| 4℃ | ∞ |
(6.5) purification of ligation product
Placing DNA Selection Beads magnetic beads at room temperature for more than 30min, homogenizing by vortex vibration, preparing 80% ethanol solution with DEPC water, and standing at room temperature for use.
Sucking 30 mu L DNA Selection Beads magnetic beads into the reaction liquid product, blowing and mixing by a pipette, and standing for 5min at room temperature.
And (5) placing the PCR tube on a magnetic rack after instantaneous separation, standing for 5min, sucking the supernatant, and discarding.
The PCR tube was kept on a magnetic rack all the time, and 200. Mu.L of freshly prepared 80% alcohol was added for rinsing, and after 30s, the supernatant was aspirated and discarded.
The previous step was repeated for a total of 2 rinses.
The PCR was removed from the magnet holder, 26. Mu.L of DEPC water was added, and after mixing by pipetting, it was allowed to stand at room temperature for 5min. The PCR tube was immediately removed and placed on a magnetic rack for 3min, and 25. Mu.L of supernatant was aspirated into the PCR tube for a new round of purification.
Sucking 20 mu L DNA Selection Beads magnetic beads into the reaction liquid of the previous step, blowing and mixing uniformly by using a liquid transfer device, and incubating for 5min at room temperature.
And (3) placing the PCR tube on a magnetic rack after instantaneous separation, standing for 5min at room temperature, sucking the supernatant and discarding.
The PCR tube was kept on a magnetic rack all the time, and 200. Mu.L of freshly prepared 80% alcohol was added for rinsing, and after 30s, the supernatant was aspirated and discarded.
The previous step was repeated for a total of 2 rinses.
Residual liquid is sucked by a pipette, and the magnetic beads are dried at room temperature after uncapping.
The PCR tube was removed from the magnet holder, 12. Mu.L of DEPC water was added, and the mixture was stirred and incubated for 5min at room temperature.
The PCR tube was immediately detached, placed on a magnetic rack, allowed to stand for 5min, and 10. Mu.L of the supernatant was aspirated into the new PCR tube for the next reaction.
(6.6) reaction solution for amplifying library configuration library, the system is as follows:
| reagent name | Volume (mu L) |
| Reaction liquid in the last step | 10 |
| 2xSuper CanceR II High-Fidelity Mix | 12.5 |
| Primer Mix for MGIR | 2.5 |
| Total | 25 |
And (5) blowing and uniformly mixing by using a liquid transfer device, and instantaneously separating.
The PCR tube was placed in a PCR instrument and library amplification was performed as follows:
| temperature (temperature) | Time | Cycle number |
| 98℃ | 1min | 1 |
| 98℃ | 10s | 12 |
| 60℃ | 30s | 12 |
| 72℃ | 30s | 12 |
| 72℃ | 5min | 1 |
| 4℃ | ∞ | - |
(6.7) library purification
Placing DNA Selection Beads magnetic beads at room temperature for more than 30min, homogenizing by vortex vibration, preparing 80% ethanol solution with DEPC water, and standing at room temperature for use.
Sucking 22.5 mu L DNA Selection Beads magnetic beads into the amplified products of the library, blowing and mixing uniformly, and incubating for 5min at room temperature.
And (3) placing the PCR tube on a magnetic rack after instantaneous separation, standing for 5min at room temperature, sucking the supernatant and discarding.
The PCR tube was kept on a magnetic rack all the time, and 200. Mu.L of freshly prepared 80% alcohol was added for rinsing, and after 30s, the supernatant was aspirated and discarded.
The previous step was repeated for a total of 2 rinses.
The PCR tube is always placed on a magnetic frame, and the magnetic beads are uncapped and dried for about 5min.
The PCR tube was removed from the magnetic rack, 11. Mu.L of DEPC water was added, and the mixture was stirred with a pipette and allowed to stand at room temperature for 5 minutes.
The PCR tube was immediately detached, placed on a magnetic rack, allowed to stand for 5min, and 10. Mu.L of supernatant was aspirated into the new PCR tube.
(6.8) RNA library quality inspection
The detection of fragment size of the library is carried out by using a Qsep100 nucleic acid protein analyzer, and the specific operation steps are as follows:
diluting 10x of the partition Buffer to 1x with DEPC water, wherein Separation Buffer is not diluted, and adding 20 μl of mineral oil seal into 30 μl of Alignment Marker; size Marker volume 30. Mu.L, add 20. Mu.L mineral oil.
The library samples were adjusted to an optimal concentration range of 1-2 ng/. Mu.L using a 1 Xdilution Buffer, and were added to a 0.2mL PCR tube in a volume of 15-20. Mu.L.
P (Park) is a glue spraying groove, W (Wash) and C (Clean) positions are cleaning grooves, pure water is added to all three holes, and Separation Buffer is added to an S groove.
Detection was performed using an S2 cardholder.
The concentration difference of the samples is detected by a fluorescence detection method by using a Qubit fluorometer, and the concentration data of each sample is output.
7. Sequencing on machine
The transcriptome libraries constructed in this study were all sequenced using an MGISEQ-2000 sequencer using the MGISEQ-2000RS high throughput sequencing kit (PE 100).
(7.1) double Barcode cyclization
The method is carried out by using a double-Barcode cyclization kit, and comprises the following specific steps:
the library samples were mixed and put into the sample according to the distribution of the data amount, and the data amount of each lane was the same and put into the sample.
Denaturation, which is the step of denaturing a double-stranded library into a single-stranded library, the reaction procedure is as follows:
| temperature (temperature) | Time |
| Thermal cover 105 DEG C | On |
| 95℃ | 3 min |
After the reaction was completed, the PCR tube was incubated on ice for 2min.
Single strand cyclization was carried out by preparing the following reaction solution and following the procedure:
| reagent name | System (mu L) |
| Dual Barcode Splint Buffer | 11.6 |
| DNA Rapid Ligase | 0.5 |
| Total | 12.1 |
The reaction conditions were as follows:
| temperature (temperature) | Time |
| Thermal cover 105 DEG C | On |
| 37℃ | 30 min |
| 4℃ | ∞ |
The following reaction solutions were prepared by digestion with enzymes, and the following procedures were followed:
| reagent name | Volume (mu L) |
| Digestion Buffer | 1.4 |
| Digestion Enzyme | 2.6 |
| Total | 4 |
The reaction conditions were as follows:
| temperature (temperature) | Time |
| Thermal cover 105 DEG C | On |
| 37℃ | 30 min |
| 4℃ | ∞ |
The cyclization product was purified using DNA Clean Beads.
Quality inspection of cyclization products: the measurement was performed using a qubit2.0 fluorescence quantitative instrument.
(7.2) DNB production
Each MGISEQ-2000RS sequencing slide contains 4 lanes, each lane on a slide must be the same DNB, and each lane requires 50. Mu.L DNB. The preparation steps of DNB are as follows:
the amount of ssDNA library input required for each DNB preparation system was calculated as v=60 fmol/C.
The reaction solution of the following system was prepared on ice:
| reagent name | Volume (mu L) |
| TE buffer | 20 - V |
| DNB preparation buffer | 20 |
| Library ssDNA | V |
| Total | 40 |
The reaction mixture is evenly mixed by the vortex oscillator, the centrifugal machine is instantaneously separated, and then the mixture is placed in a PCR instrument for the following procedures:
| temperature (temperature) | Time |
| Thermal cover 105 DEG C | On |
| 95℃ | 1 min |
| 65℃ | 1 min |
| 40℃ | 1 min |
| 4℃ | ∞ |
Taking out DNB polymerase mixed solution II (LC), placing on ice, instantly separating by a centrifugal machine, placing on ice for standby, and blowing and mixing before use.
The following components are added into the reaction liquid:
| reagent name | Volume (mu L) |
| Reaction liquid in the last step | 40 |
| DNB polymerase Mixed solution I | 40 |
| DNB polymerase Mixed solution II (LC) | 4 |
| Total | 84 |
The reaction mixture is evenly mixed by a vortex oscillator, the centrifugal machine is instantaneously separated, and then the reaction mixture is placed in a PCR instrument for the following procedures:
| temperature (temperature) | Time |
| Thermal cover 35 DEG C | On |
| 30℃ | 25 min |
| 4℃ | ∞ |
Immediately after the temperature of the PCR instrument reached 4 ℃, 20. Mu.L of DNB stop buffer was added and gently swirled with a wide-mouth tip to mix.
After DNB preparation is completed, 2 mu L of DNB is taken, and a Qubit ssDNA Assay Kit and Qubit2.0 fluorescence quantitative instrument is used for concentration detection, wherein the concentration is more than or equal to 8 ng/. Mu.L, and the qualification is obtained.
(7.3) Loading DNB
The sequencing slide color box was removed from the-20 ℃ refrigerator and left to stand at room temperature for 60 minutes to 24 hours.
The DNB loading system was configured in a 0.2ml EP tube as follows:
| reagent name | Volume (mu L) |
| DNB Loading buffer II | 16 |
| DNB polymerase Mixed solution II (LC) | 0.5 |
| DNB | 50 |
| Total | 66.5 |
The DNB loading system is slowly and uniformly mixed by a wide-mouth suction head, then is loaded into a sequencing slide, and is placed for 30 minutes at room temperature, so that the DNB loading system can be transferred to a sequencer for use.
(7.4) preparation of sequencing reagent tank
Taking out the sequencing reagent tank, thawing in normal temperature water bath, and placing in a refrigerator at 4 ℃ for standby.
And cleaning condensed water at the cover plate and the hole site by using dust-free paper.
2.4mL of the dNTPs mixture was added to a fresh 15mL sterilization tube, then 2.4mL of the DNA polymerase mixture was added to the dNTPs mixture in the tube, and the mixture was gently mixed by inversion, and the whole mixture was added to well No. 1.
2.1mL of dNTPs mixture II is added into a new 15mL sterilizing tube, then 2.1mL of DNA polymerase mixture is added into dNTPs mixture II in the tube, the mixture is gently inverted and mixed uniformly, and then the mixture is added into the number 2 hole entirely.
And sealing the sample adding holes No.1 and No. 2 by using a matched transparent sealing film.
The 500 mu LMDA polymerase mixed solution is removed by a 1mL pipettor and added into a reagent tube of the MDA reagent, the mixture is inverted and mixed uniformly, and then the mixed solution is added into a 15 # hole, and no bubbles are ensured at the bottom of the tube during the addition.
And taking out the working solution of the cPAS AD153 barcode primer 3 in the cPAS barcode primer 3 kit, and adding 2.90mL into the No. 4 hole site to ensure that no bubble exists at the bottom of the hole site.
After all the reagents are added, the reagents are uniformly mixed, and the preparation work before the sequencing reagent tank is started is finished.
8. Library on-machine detection
A library of successful quality tests was sequenced using the MGISEQ-2000 using the PE100 program. After the sequencing results are completed, the analysis is performed by using a bioinformatics means.
9. Analysis of results
Purified RNA after removal of ribosomal RNA was detected using Qsep100 instrument, and peaks representing 18S rRNA and 28S rRNA disappeared due to the rRNA removal process (FIG. 2). After analysis of the sequencing results, it was found that the residual amount of ribosomal RNA was only about 2% after the probe of the present invention was used for library construction, and the residual rate of ribosomal RNA of the sample after the probe treatment in the commercial product was about 70%, which indicates that the probe of the present invention has excellent effect of removing chicken ribosomal RNA (FIG. 3).
In addition to the implementations described above, other implementations of the invention are possible. All technical schemes formed by equivalent substitution or equivalent transformation fall within the protection scope of the invention.
Claims (5)
1. A specific probe group combined with chicken ribosomal RNA consists of 145 antisense probes, and the sequences of the antisense probes are SEQ ID NO.1-SEQ ID NO.145.
2. Use of a set of specific probes binding to chicken ribosomal RNAs according to claim 1, characterized in that: for removing ribosomal RNA in chicken total RNA, comprising the steps of:
firstly, extracting total RNA of chicken, and combining the probe set according to claim 1 and target RNA by a gradient cooling method to form RNA-DNA hybrid double chains;
secondly, removing target RNA in the target RNA-DNA hybrid double strand by adopting RNase H enzyme;
thirdly, removing the genome DNA and the probe by DNase I enzyme;
and fourthly, recovering the purified RNA by using magnetic beads.
3. Use of a set of specific probes binding to chicken ribosomal RNAs according to claim 2, characterized in that: the hybridization system in the first step is hybridization Buffer 3 mu L, probe 1 mu L, total RNA 1 mu g, total reaction volume 15 mu L, reaction condition is 105 ℃ heat cover, 95 ℃ 2min, 95 ℃ to 22 ℃ 0.1 ℃/s,22 ℃ 5min, and the reaction is finished to 4 ℃.
4. Use of a set of specific probes binding to chicken ribosomal RNAs according to claim 2, characterized in that: the reaction system of RNase H enzyme treatment in the second step is as follows: 3 mu L of RNase H Buffer, 2 mu L of RNase H, 15 mu L of the product of the previous step and 20 mu L of the total volume; the reaction conditions were 37℃for 30min.
5. Use of a set of specific probes binding to chicken ribosomal RNAs according to claim 2, characterized in that: the reaction system of DNase I enzyme treatment in the third step is as follows: DNase I Buffer 27.5 mu L, DNase I2.5 mu L, the product of the last step 20 mu L, total volume 50 mu L; the reaction conditions were 37℃for 30min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310713150.6A CN116463398B (en) | 2023-06-16 | 2023-06-16 | Specific probe set combined with chicken ribosomal RNA and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310713150.6A CN116463398B (en) | 2023-06-16 | 2023-06-16 | Specific probe set combined with chicken ribosomal RNA and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116463398A CN116463398A (en) | 2023-07-21 |
| CN116463398B true CN116463398B (en) | 2023-10-03 |
Family
ID=87181119
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310713150.6A Active CN116463398B (en) | 2023-06-16 | 2023-06-16 | Specific probe set combined with chicken ribosomal RNA and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116463398B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106319639A (en) * | 2015-06-17 | 2017-01-11 | 深圳华大基因科技有限公司 | Sequencing library construction method and device |
| CN111518870A (en) * | 2020-02-04 | 2020-08-11 | 广东美格基因科技有限公司 | Reverse transcription primer pool and kit for removing ribosomal RNA and method for removing ribosomal RNA |
| CN112176031A (en) * | 2020-09-18 | 2021-01-05 | 上海英基生物科技有限公司 | Construction method and kit of enucleated ribosome RNA sequencing library |
| CN113061646A (en) * | 2020-01-02 | 2021-07-02 | 深圳华大基因科技服务有限公司 | Method for preparing single-chain length probe, method for removing ribosomal RNA and kit |
| CN113652469A (en) * | 2021-08-17 | 2021-11-16 | 上海市肺科医院 | Method and kit for removing ribosomal RNA and/or mitochondrial RNA in sample |
-
2023
- 2023-06-16 CN CN202310713150.6A patent/CN116463398B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106319639A (en) * | 2015-06-17 | 2017-01-11 | 深圳华大基因科技有限公司 | Sequencing library construction method and device |
| CN113061646A (en) * | 2020-01-02 | 2021-07-02 | 深圳华大基因科技服务有限公司 | Method for preparing single-chain length probe, method for removing ribosomal RNA and kit |
| CN111518870A (en) * | 2020-02-04 | 2020-08-11 | 广东美格基因科技有限公司 | Reverse transcription primer pool and kit for removing ribosomal RNA and method for removing ribosomal RNA |
| CN112176031A (en) * | 2020-09-18 | 2021-01-05 | 上海英基生物科技有限公司 | Construction method and kit of enucleated ribosome RNA sequencing library |
| CN113652469A (en) * | 2021-08-17 | 2021-11-16 | 上海市肺科医院 | Method and kit for removing ribosomal RNA and/or mitochondrial RNA in sample |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116463398A (en) | 2023-07-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20160122753A1 (en) | High-throughput rna-seq | |
| JP2002507121A (en) | RNA isolation method | |
| CN108611398A (en) | Genotyping is carried out by new-generation sequencing | |
| CN111321208B (en) | Library construction method based on high-throughput sequencing | |
| CN107893100A (en) | A kind of unicellular mRNA reverse transcriptions and the method for amplification | |
| CN111378720A (en) | Construction method and application of sequencing library of long-chain non-coding RNA | |
| CN112410331A (en) | Linker with molecular label and sample label and single-chain library building method thereof | |
| CN110592200B (en) | Multiplex PCR method for improving amplification specificity and uniformity | |
| CN109337956B (en) | Design method and kit for enriching multi-gene stem-loop probe based on NGS technology | |
| CN110205365B (en) | High-throughput sequencing method for efficiently researching RNA interaction group and application thereof | |
| CN116463398B (en) | Specific probe set combined with chicken ribosomal RNA and application thereof | |
| CN117089653A (en) | Compositions for RNA virus detection based on high throughput amplicon sequencing | |
| CN111979353A (en) | Library construction method for sequencing novel coronavirus SARS-CoV-2 full-length genome | |
| CN111501106A (en) | Construction method, device and application of high-throughput sequencing library of exosome RNA | |
| WO2023202030A1 (en) | Method for constructing high-throughput sequencing library of small rna | |
| CN116377099A (en) | Primer, kit and method for detecting fever with hemorrhagic disease pathogen | |
| CN111057747A (en) | Method for extracting microbial nucleic acid with host genome DNA removing function and kit | |
| CN116287357A (en) | Respiratory tract pathogenic bacteria detection kit based on targeted amplicon sequencing | |
| CN110804655B (en) | Absolute quantification method for metagenome | |
| CN112522792A (en) | Construction method of RNA sequencing library | |
| CN106283198B (en) | Library construction method for single cell whole genome bisulfite sequencing | |
| CN112063615A (en) | Genome DNA extracting solution, genome DNA extracting method and application thereof | |
| CN114657239B (en) | Primer combination, kit and library construction method for multiplex PCR second generation sequencing | |
| CN210656930U (en) | Sequencing library construction unit of long-chain non-coding RNA and system for sequencing long-chain non-coding RNA | |
| CN117165657A (en) | Construction method of RNA library |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |