CN105779439A - Library construction method for RNA 5'-terminal information acquired through low-initial-dose high-throughput sequencing analysis transcription - Google Patents
Library construction method for RNA 5'-terminal information acquired through low-initial-dose high-throughput sequencing analysis transcription Download PDFInfo
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Abstract
The invention belongs to the technical field of genomics, and particularly relates to a micro CAGE-seq library construction method through which 5' CAP sequence information can be specifically acquired. Through the combined effect of T4 PNK and TEX, RNA without a 5' CAP structure at the 5' terminal in total RNA is eliminated, and all RNA with a 5' CAP structure is retained; a corresponding RT primer and a corresponding TS primer are designed, and RNA 5'-terminal information is enriched in the reverse transcription and template replacement processes; a sequencing primer and a barcode sequence are introduced through PCR. By means of the method, a tedious half-inhibiting PCR process is omitted, library construction time is shortened, and experiment procedures are simplified; the initial dose of RNA is low and is about 100 ng-1 microgram; the method is a powerful mode for studying gene expression and is suitable for studying gene regulatory networks.
Description
Technical field
The invention belongs to biology field, acquisition is transcribed in the high-flux sequence analysis particularly to a kind of low initial amount
The library constructing method of RNA 5 ' client information.
Background technology
The topmost mode of expression regulation of gene is transcriptional control, i.e. promoter cis acting element is mutual with transcriptional machinery
The regulation and control made.Be positioned at that the core promoter of transcriptional start site (TSS) upstream then contains with transcriptional machinery interaction is main
Functional element, therefore the precise Identification of promoter to research gene expression regulation most important.Meanwhile, different transcription initiations
Site, can cause gene to use 5 ' different UTR.5 ' UTR are most important to the translation initiation of mRNA, are translation efficiency regulation and control
One of main targeting regions.Most genes have a transcriptional start site of more than 2, therefore system identification mRNA turn
Record initiation site, on the one hand can identify core promoter element, on the other hand may determine that the 5 ' UTR of mRNA, is research base
Because of the powerful way expressed, it is suitable for and studies with gene regulatory network.
People study 5 ' UTR sequence information the earliest, be employing 5 ' RACE method (B.C.Schaefer Volume 227,
Issue 2, May 1995, Pages 255 273), but flux is relatively low, builds clone time-consuming long.1st high flux obtains and turns
Record initiation site Forecasting Methodology is that gene expression method (CAGE) analyzed by cap, and the method is by Sanger sequencing development the earliest
Come, and complete RNA cap sequence can be obtained by cDNA clone.Although the prediction of transcriptional start site is had by the method
Effect, but needs a large amount of high-quality RNA, it is thus achieved that transcriptional start site the shortest, be only 20～21 length of nucleotides.
Being developed by high-throughout secondary DNA sequencing technology (NGS), CAGE method is improved, it has been investigated that, pass through
The transcriptional start site that the method is complicated in can obtaining whole genome range is distributed and unique promoter.Therefore, CAGE
Derived after being combined with RNA sequencing technologies the DeepCAGE method based on CAGE strategy, CAGEscan method (such as Fig. 1),
Nano-CAGE method (such as Fig. 2) and PEAT method.So far the method analyzing transcriptional start site based on Sanger checks order
Receiving the challenge that RNA sequencing technologies is combined with CAGE, such as, PEAT method and the double end sequencing of CAGEscan method can obtain and turn
Record initiation site maps and transcriptional start site downstream is interval, has good connectedness and can promote to identify special transcribing
This；Additionally, double end sequencings alleviate the check and correction to single short reading duplicate block, the weight of sequence can be obtained by RNA order-checking
Multiple characteristic；Nano-CAGE method solves CAGE method needs the shortcoming of a large amount of RNA, can pass through the amplifying technique total serum IgE from 10ng
Amount obtains the mapping of transcriptional start site；Its semi-inhibit PCR method is effectively reduced short fragment amplification.Although these methods are tied
Having closed NGS technology, overcome the deficiency of some CAGE methods, but there is certain drawback, such as, qRT-PCR to be carried out determines
Amplification cycles number, carry out time-consumingly the longest semi-inhibit PCR process；Operating procedure during detection amplification, Ke Nengying
Ring the frequency that transcriptional start site occurs；Complex steps, it is difficult to operation etc..
Summary of the invention
In order to solve the deficiencies in the prior art, the present invention is changed on the basis of existing nano-CAGE method
Enter, it is provided that a kind of energy specificity obtains the trace CAGE-seq library constructing method of 5 ' end cap minor structure sequence (5 ' CAP) information.
Use T4 polynueleotide kinase (T4Polynucleotide Kinase, T4 PNK), and TerminatorTM5′-
Phosphate-Dependent Exonuclease (TEX enzyme) (Epicentre company, article No.: TER51020) is used in combination,
Eliminate without 5 ' CAP rRNA and the pollution of the RNA of degraded, the specific enrichment RNA containing 5 ' CAP；The present invention redesigns RT
The sequence of primer and TS primer (template switching oligonucleotide, TS oligo), adds circumscribed
Nuclease I (ExonucleaseI, Fermentas, article No.: EN5081) clears up residue mononucleotide chain TS primer and RT primer
Step, it is to avoid loaded down with trivial details semi-inhibit PCR process, shortens and builds the storehouse time, simplify experiment flow.
The present invention provides a kind of method building CAGE-seq library, and it comprises the steps:
1) RNA sample is processed with DNaseI (promega), it is thus achieved that without the RNA of DNA pollution；
2) 5 ' ends are removed without the RNA of cap sequence: use T4Polynucleotide Kinase (NEB, article No.:
M0201V), Terminator will be re-used without 5 ' the end phosphorylations of the RNA of 5 ' end cap minor structuresTM5′-Phosphate-
Dependent Exonuclease (Epicentre) digests the RNA of above-mentioned 5 ' end phosphorylations, retains with 5 ' end caps
The RNA of minor structure；
3) synthesis cDNA:RT primer (RT-Primer) hybridizes to RNA, at PrimeScriptTM Reverse
Start polyreaction under the effect of Transcriptase (TAKARA, article No. 2680A), form Article 1 cDNA chain, when being aggregated to
With the 5 ' ends of the RNA of 5 ' end cap minor structures, 3 cytosine of continuous print will be synthesized more, meanwhile these three cytosine and
3 ' 3 the guanine complementary pairings held of TS primer (template switching oligonucleotide, TS-oligo)
In conjunction with, under the effect of polymerase, initially form Article 2 chain cDNA；ExonucleaseI (NEB) clears up residue mononucleotide
Chain TS primer and RT primer, it is to avoid joint is from the amplification connected；The sequence of described RT primer as shown in SEQ NO.1, TS primer
Sequence is as shown in SEQ NO.2；
4) PCR amplification: to step 3) the most synthetic cDNA, carry out PCR amplification, introduce sequencing primer and corresponding bar shaped
Code sequence, obtains complete trace CAGE-seq sequencing library；Described PCR forward primer sequence as shown in SEQ NO.3, PCR
Reverse primer sequences is as shown in SEQ NO.4.
Sequencing library Illumina Nextseq 500 sequenator that said method obtains is carried out both-end order-checking, both-end
Survey 150 bases.
The present invention compared with prior art, has the following advantages:
1, T4PNK enzyme and TerminatorTM5 '-Phosphate-Dependent Exonuclease are used in combination, special
Property digest the RNA without 5 ' cap structures, remain the RNA containing 5 ' cap, make the purposiveness of research and specificity higher.
2, without doing semi-inhibit PCR, shortening and build the storehouse time, make operating process simpler, cost is lower.
3, the TS primer of our design, with the addition of 6 randomized bases before 3 rG bases, and when adding order-checking, base is put down
Weighing apparatus property.
Accompanying drawing explanation
Fig. 1 CAGEscan library construction flow process.
Fig. 2 nano-CAGE library Library development flow figure.
The trace CAGE-seq library Library development flow figure of Fig. 3 present invention.
Fig. 4 trace CAGE library screening product electrophoretogram
Totally 14 swimming lanes: 1 and 11 is D2000Marker；2 be the 50bp ladder, 3-6 of Tian Gen company be library screening
Product fragment two；7-10 is library screening product fragment three；10-14 is library screening product fragment four.
Fig. 5 compares (Md Salimullah, Sakai Mizuho, Charles Plessy, and with delivering data in literature
Piero Carninci, Cold Spring Harb Protoc.；2011(1):pdb.prot5559.)
A is nano-CAGE data profile 5 ' UTR ratio 38.58%；B is trace CAGE data profile 5 ' UTR ratio
30.76%；Our experimental result with deliver data, be all enriched with in 5 ' UTR districts, it was demonstrated that the trace CAGE-of the present invention
Seq library banking process is effective.
Detailed description of the invention
By combination accompanying drawing described further below it will be further appreciated that the features and advantages of the invention.The enforcement provided
Example is only the explanation to the inventive method, and limits remaining content that the present invention discloses never in any form.
Relevant DNA and RNA basic operation in all embodiments is all with reference to " molecular cloning: LABORATORY MANUAL " (gold
Winter wild goose etc. is translated, Science Press, Beijing (1998)) and " fine works Molecular Biology " (Yan Ziyi, Science Press, Beijing
(1998)).Enzyme used in molecule manipulation and special reagent bracket later have all indicated corresponding company.Following example
Using Hela cell as experiment material.
[embodiment 1] removes the 5 ' ends RNA without 5 ' cap
Taking 200ng Total RNA, volume is less than or equal to 10 μ l, adds 1 μ l DnaseI enzyme (promega) and clears up RNA
DNA in sample, prevents genome pollution.RNA after purification, uses T4 PNK (NEB) and TEX enzyme (TerminatorTM5′-
Phosphate-Dependent Exonuclease) (Epicentre), clear up the RNA without 5 ' cap.
1.1.T4PNK enzyme reaction system (making 5 ' the end phosphorylations of the RNA without 5 ' cap):
Gnomegen company Gnome size selector is used to be purified sample.Finally by sample back dissolving to 16.5 μ
l。
1.2.TEX(TerminatorTM5 '-Phosphate-Dependent Exonuclease) reaction system (digests
All 5 ' ends do not have the RNA of cap):
Gnomegen company Gnome size selector is used to be purified sample, finally by sample back dissolving to 5 μ l.
[embodiment 2] RT and TS primer synthesis cDNA.
Carrying out reverse transcription synthesis cDNA with PrimeScript (TAKARA), concrete steps provide with reference to nano-CAGE document
Material.(Md Salimullah, Sakai Mizuho, Charles Plessy, and Piero Carninci, Cold Spring
Harb Protoc.；2011(1):pdb.prot5559.)
Configuration mixA
Sorbitol/Trehalose stock (sorbitol/trehalose mixed liquor) configuration provides with reference to nano-CAGE document
Material.Configure following reagent solution, all use the water configuration of free nucleic acid hydrolytic enzyme.
1.D-sorbitol (Wako 198-03755) (0.89g/mL [4.9M]), trehalose (Fluka/Sigma-Aldrich
90208) (0.727g/mL) 121 DEG C of autoclaving 30min.
2. add the saturated aqueous trehalose of 5mL in 10mL of 4.9M D-glucitol solution.
3. add with suction pipe and < in the resin 100 of 1cm to 15mL centrifuge tube, mix.Room temperature (25 DEG C) stands 3 hours,
2000rpm is centrifuged 10min.
4. supernatant is transferred in new centrifuge tube, 20 DEG C of preservations.
RT primer sequence (takara synthesis):
5 '-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNN3 ' TS primer (template switched
Oligonucleotide) sequence (takara synthesis): (underscore is 3 RNA base guanine):
5’-CTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNNrGrGrG 3’
Configuration mix B
After having reacted, add 1 μ l ExonucleaseI and digest primer, above-mentioned product will add 36 μ l
Gnome Size Selector is purified sample, with 20 μ l DEPC water elution samples, 20 μ l supernatants is proceeded to new PCR pipe
In, it is thus achieved that cDNA.
[embodiment 3] PCR amplification and fragment are screened
In 50 μ l reaction systems, containing the cDNA template obtained in 20 μ l embodiments 2,1 times of concentration gnomegen company
HiFi buffer, the forward primer of 25pmol and the reverse primer of 25pmol, supply volume with water.The parameter of thermal cycle is such as
Under: 98 DEG C of for 45 seconds；Of10-15:98 DEG C of for of PCR cycle 15 seconds, 60 DEG C of for 30 seconds, 72 DEG C of for 30 seconds；72℃
1min；4 DEG C of insulations.
PCR forward primer sequence (takara):
AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGPCR reverse primer sequences
(takara): (underscore is barcode sequence):
CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACG
Add in 80 μ l Gnome Size Selector to 50 μ l reactant liquors, inhale and play mixing 10 times, after standing 5min, put
In magnetic frame 3min, absorption supernatant to new PCR pipe, it is careful not to be drawn onto magnetic bead, is added to 20 μ l Gnome Size
In Selector, inhale and play mixing 10 times, after standing 5min, be put in 3min on magnetic frame, abandon supernatant, add 200 μ l 70%
3 cleanings are played in Ethanol suction.70%Ethanol is blotted only, sample is taken off from magnetic frame, be dried 5min.Each
Sample takes 20 μ l DEPC water back dissolvings, and 5 resuspended magnetic beads are made a call in suction.Rotary sample 2min at a slow speed, then stands on magnetic frame
3min.20 μ l supernatants are proceeded in new EP pipe.The concentration in library is measured with Qubit luminoscope (Invitrogen).With
Illumina company Next500 sequenator carries out both-end order-checking, and often end measures 150 bases.
SEQUENCE LISTING
<110>Wuhan beauty of life Science and Technology Ltd.
<120>library constructing method of RNA 5' client information of acquisition is transcribed in the high-flux sequence analysis of a kind of low initial amount
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 40
<212> DNA
<213>synthetic RT primer
<220>
<221> misc_feature
<222> (35)..(40)
<223> n is a, c, g, or t
<400> 1
gtgactggag ttcagacgtg tgctcttccg atctnnnnnn 40
<210> 2
<211> 42
<212> DNA
<213>synthetic TS primer
<220>
<221> misc_feature
<222> (31)..(36)
<223>n is a, c, g, or t, rg are RNA base guanine
<400> 2
ctctttccct acacgacgct cttccgatct nnnnnnrgrgrg 39
<210> 3
<211> 46
<212> DNA
<213>synthetic PCR forward primer sequence
<400> 3
aatgatacgg cgaccaccga gatctacact ctttccctac acgacg 46
<210> 4
<211> 48
<212> DNA
<213>synthetic PCR reverse primer sequences
<400> 4
caagcagaag acggcatacg agatcgtgat gtgactggag ttcagacg 48
Claims (2)
1. the method building CAGE-seq library, it is characterised in that comprise the steps:
1) RNA sample is processed with DNaseI, it is thus achieved that without the RNA of DNA pollution；
2) 5 ' ends RNA without cap sequence is removed: use T4 polynueleotide kinase, will be without the RNA's of 5 ' end cap minor structures
5 ' end phosphorylations, re-use the Terminator 5-Phosphate-Dependent of Epicentre company
Exonuclease digests the RNA of above-mentioned 5 ' phosphorylations, retains the RNA with 5 ' end cap minor structures；
3) synthesis cDNA:RT primer hybridization is to RNA, starts polyreaction, form Article 1 cNDA chain under the effect of enzyme, when poly-
Close the 5 ' ends of the RNA with 5 ' end cap minor structures, can many synthesis continuous print 3 cytosine, meanwhile these three cytosine
Under the effect of polymerase, Article 2 chain can be initially formed combined with 3 guanine complementary pairings that the 3 ' of TS primer are held
cDNA；The exonuclease I of Fermentas company clears up residue mononucleotide chain TS primer and RT primer；Described RT primer
Sequence as shown in SEQ NO.1, the sequence of TS primer is as shown in SEQ NO.2；
4) PCR amplification: and the cDNA of purification the most synthetic to step 3), carries out PCR amplification, introduces sequencing primer with corresponding
Bar code sequence, obtain complete trace CAGE-seq sequencing library；Described PCR forward primer sequence such as SEQ NO.3 institute
Showing, PCR reverse primer sequences is as shown in SEQ NO.4.
2. a CAGE-seq method, it is characterised in that the sequencing library Illumina company that the method for claim 1 builds
Hiseq3000/2500 or Nextseq500 sequenator carries out both-end order-checking, and both-end surveys 150 bases.
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Cited By (3)
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| CN109750031A (en) * | 2019-02-22 | 2019-05-14 | 河南师范大学 | Using the library constructing method of high throughput sequencing technologies detection transcription initiation site |
| CN111051524A (en) * | 2018-03-22 | 2020-04-21 | 伊鲁米纳公司 | Preparation of nucleic acid libraries from RNA and DNA |
| CN111635930A (en) * | 2020-05-12 | 2020-09-08 | 中国农业科学院农业基因组研究所 | Method for high-throughput sequencing and calling full-length sequence of unknown RNA |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111051524A (en) * | 2018-03-22 | 2020-04-21 | 伊鲁米纳公司 | Preparation of nucleic acid libraries from RNA and DNA |
| CN109750031A (en) * | 2019-02-22 | 2019-05-14 | 河南师范大学 | Using the library constructing method of high throughput sequencing technologies detection transcription initiation site |
| CN111635930A (en) * | 2020-05-12 | 2020-09-08 | 中国农业科学院农业基因组研究所 | Method for high-throughput sequencing and calling full-length sequence of unknown RNA |
| CN111635930B (en) * | 2020-05-12 | 2023-10-24 | 中国农业科学院农业基因组研究所 | Method for extracting unknown RNA full-length sequence by high-throughput sequencing |
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