CN110499356A - A kind of construction method of the sequencing library of RNA with poly(A) tail in sample to be tested - Google Patents
A kind of construction method of the sequencing library of RNA with poly(A) tail in sample to be tested Download PDFInfo
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Abstract
The invention discloses a kind of construction methods of the sequencing library of the RNA in sample to be tested with poly (A) tail.This method comprises the following steps: the total serum IgE of sample to be tested is taken, end extension is carried out using guidance primer, is then sufficiently digested with USER enzyme, the nucleic acid fragment that recycling length is 200nt or more；Nucleic acid fragment is subjected to reverse transcription, then template exchange is carried out with TSO, obtains cDNA；The cDNA is taken, PCR amplification is carried out using PCR primer, obtains amplification cDNA；The amplification cDNA is taken, sequencing library is constructed, finally upper machine sequencing.It is demonstrated experimentally that can analyze the sequence of the RNA with poly (A) tail, the sequence including poly (A) tail using the sequencing library that method provided by the invention constructs.The present invention has important application value for the research of poly (A) tail of RNA.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to the survey of the RNA in sample to be tested with poly (A) tail a kind of
The construction method in preface library.
Background technique
Most of mRNA (mRNA) and long-chain non-coding RNA (long intergenic non-coding RNA,
) etc. lincRNA mature rnas usually will receive numerous posttranscriptional modifications regulations.It is most of under the catalysis of poly (A) polymerase
MRNA and lincRNA transcription while be coupled with poly (A) tail without template.Poly (A) tail is considered as adjusting
Control one of rna stability and the key factor of transcriptional efficiency.MRNA and lincRNA with poly (A) are only accounted in RNA intracellular
Sub-fraction.The affinity purification that Oligo (dT) is relied on is the common method for separating mRNA, but this method is for longer poly
(A) tail has inevitable Preference.Thus, when analyzing poly (A) tail, need to avoid poly (A) as far as possible
Skewed popularity be enriched with to obtain original true poly (A) information.
Into high-flux sequence (next generation sequencing, NGS) after the epoch, transcript profile correlative study
It emerges one after another.But the base recognizer of existing NGS Technology application can not handle the homopolymerization sequence for being longer than 30nt, poly (A) tail
Bar specific base composition also fail to be illustrated well.Even Sanger PCR sequencing PCR is also difficult to identify longer homopolymerization sequence
Column.Smart-seq2 is a kind of extremely sensitive unicellular RNA-seq technology, which can pass through the anchor primer of 3 ' UTR
5 '-AAGCAGTGGTATCAACGCAGAGTACT30VN-3 ' (N represents any base, and V represents A, C or G) carry out reverse transcription and
The building of cDNA library.The N and V of the prime end can be anchored to the end of 3 ' UTR, be analyzed by data from final
Poly (A) tail is removed in cDNA library, excludes the influence to sequence assembling of homopolymerization sequence.Illumina platform other
RNA-seq tool can also remove the influence of poly (A) in library construction, sequencing and data analysis process.Based on PacBio
The Iso-seq technology of platform can use the strategy similar to Smart-seq2, and text is constructed from the cDNA for rejecting poly (A)
Library.Therefore, the conventional RNA-seq Iso-seq data that Illumina/PacBio platform generates lack poly (A) information.
At present, PAL-seq (poly (A) tail length profiling by sequencing) and TAIL-
Seq can detect the length of poly (A) tail within the scope of full transcript profile by optimizing the base recognizer for poly (T).
Using these means, researchers are from yeast, cell line, mouse liver, Arabidopsis leaf, drosophila, frog and zebra fish
Poly (A) tail length dynamically some data are obtained in the materials such as embryo.The special sequencing side of one kind used by PAL-seq
Method can only be realized on a sequenator (Illumina GenomeAnalyzer II) to have stopped production.PAL-seq method
The principle for measuring poly (A) length is the dUTP for mixing dTTP and biotin labeling during primer extend, passes through reading
The mixed ratio of biotin labeling estimates poly (A) tail length.PAL-seq method cannot chemically identify from principle
Non- A residue in poly (A) tail, is also unable to get the data of single base resolution ratio.TAIL-seq and its mRNA enrichment changes
Good version mTAIL-seq needs to determine the end poly (T) using special base recognizer by analysis primitive sequencer image
Position.Primitive sequencer image needed for only a small amount of sequenator can provide the analysis at present.It is readily apparent that being based on
Existing commercialization sequencing service and general sequencing instrument, most of user can not be surveyed using above-mentioned mTAIL-seq well
Sequence method.Other two defect of this method is: the special base recognizer needed for it is long for effective reading of poly (T) only
There is 231bp, and is also not readily available the non-T residue information inside poly (A) tail.It can be seen that TAIL-seq and PAL-
Although seq provides description poly (A) length, the non-adenosine in detection end poly (A) modifies the means of (non-A is modified), and RNA tail
Uridine (uridine, U) and guanosine (guanosine, G) modification of bar 3 ' ends also have benefited from this and identified come out.However,
Base composition in poly (A) tail except 3 ' ends is not clear.
In Mouse Eggs maturation and the growth course of body early embryo, the removing including source of parents mRNA and zygotic gene has occurred
A variety of development events including group activation (ZGA), these critical events and a variety of RNA for being stored in GV egg cell are closely related,
And regulation of these RNA by its poly (A) tail.
Summary of the invention
The purpose of the present invention is obtaining the complete sequence of poly (A) tail in sample to be tested RNA, so as to more accurate
Analyze the structure and function of poly (A) tail.
The present invention protects the construction method of the sequencing library of the RNA in sample to be tested with poly (A) tail first, successively
May include following steps:
(a1) total serum IgE for taking sample to be tested carries out end extension using guidance primer, USER enzyme is then added and sufficiently disappears
Change, the nucleic acid fragment that recycling length is 200nt or more；
Guide primer from 5 ' ends to 3 ' ends successively may include DNA fragmentation first and DNA fragmentation third；
DNA fragmentation first can be the arbitrary sequence and not of 15-40bp (such as 15-25bp, 25-40bp, 15bp, 25bp or 40bp)
PCR amplification can be used in conjunction with poly (A) tail；
DNA fragmentation third can be made of 3-24bp (such as 3-18bp, 18-24bp, 3bp, 18bp, 24bp) nucleotide, Mei Gehe
Thuja acid can be dU or T；At least one in 3 nucleotide of DNA fragmentation the third 5 ' end is dU；
(a2) nucleic acid fragment is taken, reverse transcription is carried out with RT primer, obtains cDNA；
RT primer is all or part of of the nucleotide sequence of DNA fragmentation first；
(a3) cDNA is taken, carries out template exchange with TSO；
TSO from 5 ' ends to 3 ' ends successively may include DNA fragmentation fourth and nucleic acid fragment fourth；
DNA fragmentation fourth can be the arbitrary sequence of 15-40bp (such as 15-25bp, 25-40bp, 15bp, 25bp or 40bp), use
In PCR amplification；
Nucleic acid fragment fourth may include section 2, and section 2 is used for and the cDNA combines that (5 ' ends of cDNA have been when reverse transcription
Add C)；Section 2 can be by N number of guanosine ribonucleoside acid or " (N-1) a guanosine ribonucleoside acid and 1 have carried out lock core
The guanine deoxyribonucleotide of acid modification " composition, N can be 3 or more natural number；
(a4) cDNA for taking into step (a3) carries out PCR amplification using PCR primer, obtains amplification cDNA；
Upstream primer in PCR primer is all or part of of the nucleotide sequence of DNA fragmentation fourth；
Downstream primer in PCR primer is all or part of of the nucleotide sequence of DNA fragmentation first；
(a5) the amplification cDNA is taken, is sequenced；Obtain the sequencing library of the RNA in sample to be tested with poly (A) tail.
The present invention also protects the construction method of the sequencing library of the RNA in unicellular with poly (A) tail, can successively wrap
Include following steps:
(b1) it is cracked unicellular, obtains cell pyrolysis liquid；Then end extension is carried out using guidance primer；Later
USER enzyme is added sufficiently to digest, the nucleic acid fragment that recycling length is 200nt or more；
Guide primer from 5 ' ends to 3 ' ends successively may include DNA fragmentation first and DNA fragmentation third；
DNA fragmentation first can be the arbitrary sequence and not of 15-40bp (such as 15-25bp, 25-40bp, 15bp, 25bp or 40bp)
PCR amplification can be used in conjunction with poly (A) tail；
DNA fragmentation third can be made of 3-24bp (such as 3-18bp, 18-24bp, 3bp, 18bp, 24bp) nucleotide, Mei Gehe
Thuja acid is dU or T；At least one in 3 nucleotide of DNA fragmentation the third 5 ' end is dU；
(b2) nucleic acid fragment is taken, reverse transcription is carried out with RT primer, obtains cDNA；
RT primer is all or part of of the nucleotide sequence of DNA fragmentation first；
(b3) cDNA is taken, carries out template exchange with TSO；
TSO from 5 ' ends to 3 ' ends successively may include DNA fragmentation fourth and nucleic acid fragment fourth；
DNA fragmentation fourth can be the arbitrary sequence of 15-40bp (such as 15-25bp, 25-40bp, 15bp, 25bp or 40bp), use
In PCR amplification；
Nucleic acid fragment fourth may include section 2, and section 2 is used for and the cDNA combines that (5 ' ends of cDNA have been when reverse transcription
Add C)；Section 2 is by N number of guanosine ribonucleoside acid or " (N-1) a guanosine ribonucleoside acid and 1 have carried out lock nucleic acid
The guanine deoxyribonucleotide of modification " composition, N can be 3 or more natural number；
(b4) cDNA for taking into step (b3) carries out PCR amplification using PCR primer, obtains amplification cDNA；
Upstream primer in PCR primer is all or part of of the nucleotide sequence of DNA fragmentation fourth；
Downstream primer in PCR primer is all or part of of the nucleotide sequence of DNA fragmentation first；
(b5) the amplification cDNA is taken, is sequenced；Obtain the sequencing library of the RNA in unicellular with poly (A) tail.
Any of the above-described guidance primer can be successively made of from 5 ' ends to 3 ' ends DNA fragmentation first and DNA fragmentation third.
Any of the above-described TSO can be successively made of from 5 ' ends to 3 ' ends DNA fragmentation fourth and nucleic acid fragment fourth.
Any of the above-described nucleic acid fragment fourth can be specifically made of section 2.
In any of the above-described method, in step (b1), the side of " being cracked unicellular, obtain cell pyrolysis liquid "
Method can are as follows: to unicellular middle addition nonionic surface active agent and RNase inhibitor, is incubated for, obtains cell pyrolysis liquid.
The nonionic surface active agent can be TritonX-100.
In the step (b1), the method for " cracking unicellular, obtain cell pyrolysis liquid " can are as follows: in unicellular
Aqueous solution and 1 μ L RNase inhibitor that 19 μ L contain 0.2% (v/v) TritonX-100 is added, is incubated for, obtains cell pyrolysis liquid.
The parameter of the incubation can be 70-90 DEG C of (such as 70-80 DEG C, 80-90 DEG C, 70 DEG C, 80 DEG C or 90 DEG C) incubation 3-
10min (such as 3-5min, 5-10min, 3min, 5min or 10min).
Any of the above-described guidance primer further includes DNA fragmentation second, and DNA fragmentation second is located at 3 ' ends of DNA fragmentation first, DNA
5 ' ends of segment third；DNA fragmentation second can be the Barcode sequence of 8-30bp (such as 8-16bp, 16-30bp, 8bp, 16bp or 30bp)
Column, for distinguishing different sample to be tested or unicellular.
Any of the above-described guidance primer successively can be by DNA fragmentation first, DNA fragmentation second and DNA from 5 ' ends to 3 ' ends
Segment third forms.
In any of the above-described method, in step (a1) and step (b1), the progress end, which extends, also to be needed in body
DNTP and archaeal dna polymerase are added in system.
The archaeal dna polymerase concretely Klenow segment (exo-, NEB).
The condition that the end extends can are as follows: 32-42 DEG C of (such as 32-37 DEG C, 37-42 DEG C, 32 DEG C, 37 DEG C or 42 DEG C) incubation
30min-90min (such as 30min-60min, 60min-90min, 30min, 60min or 90min).
The condition of " USER enzyme sufficiently digests " can be 32-42 DEG C (such as 32-37 DEG C, 37-42 DEG C, 32 DEG C, 37 DEG C or 42
DEG C) it is incubated for 30min-90min (such as 30min-60min, 60min-90min, 30min, 60min or 90min).
In any of the above-described method, in step (a2) and step (b2), since reverse transcriptase has terminal enzyme (DNA)
Activity, so reverse transcriptase can add C in 5 ' ends of reverse transcription product (i.e. cDNA).
In any of the above-described method, in step (a3) and step (b3), the nucleic acid fragment fourth may include section 1,
Section 1 is located at 5 ' ends of section 2；The section 1 can be any of 0-10bp (such as 0-2bp, 2-10bp, 0bp, 2bp or 10bp)
Sequence.
Any of the above-described nucleic acid fragment fourth can be specifically made of section 1 and section 2.
In any of the above-described method, in the step (a3) and step (b3), the DNA fragmentation fourth can be DNA piece
Section first.
If DNA fragmentation fourth and DNA fragmentation first include DNA fragmentation oneself, upstream primer or downstream primer can be DNA fragmentation
Oneself nucleotide sequence it is all or part of, upstream primer and downstream primer can be identical at this time, i.e., are carried out using single primer
PCR amplification.
In any of the above-described method, in the step (a5) and step (b5), " taking amplification cDNA, sequencing " can are as follows:
It will be greater than the amplification cDNA of 200bp and the amplification cDNA mixing greater than 2kb, tried later using SMRTbell Template Prep
Agent box constructs the library SMRTbell Template；The library SMRTbell Template is sequenced using PacBio platform.
Using any of the above-described the method building sample to be tested in poly (A) tail RNA sequencing library or
Using any of the above-described the method building it is unicellular in poly (A) tail RNA sequencing library in RNA include
Complete poly (A) tail information.
Using any of the above-described the method building sample to be tested in poly (A) tail RNA sequencing library or
Using any of the above-described the method building it is unicellular in poly (A) tail RNA sequencing library in RNA include
Complete RNA information.
Above, the nucleotide sequence for guiding primer can be 5 '-AAGCAGTGGTATCAACGCAGAGTACTACTAGAGT
AGCACTCdUTTTTTTTTdUT TTTTTTT-3'.The nucleotide sequence of DNA fragmentation first or DNA fragmentation fourth can be 5 '-
AAGCAGTGGTATCAACGCAGAGTAC-3'.The nucleotide sequence of DNA fragmentation second can be 5 '-TACTAGAGTAGCACTC-3 '.
The nucleotide sequence of DNA fragmentation third can be 5 '-dUTTTTTTTTdUTTTTTTTT-3 '.The nucleotide sequence of RT primer can be 5 '-
AAGCAGTGGTATCAACGCAGAGTAC-3'.The nucleotide sequence of upstream primer and downstream primer can be 5 '-
AAGCAGTGGTATCAACGCAGAGT-3'.The nucleotide sequence of nucleic acid fragment fourth can be 5 '-ATrGrGG*- 3 ' (rG expression birds
Purine ribonucleotide, G*It indicates to have carried out the guanine deoxyribonucleotide that lock nucleic acid is modified).The nucleotides sequence of section 1
Column can be 5 '-AT-3 '.The nucleotide sequence of section 2 can be 5 '-rGrGG*-3'.The nucleotide sequence of TSO can be 5 '-AAGCA
GTGGTATCAACGCAGAGTACATrGrGG*-3’。
Any one of the present invention also protects S1)-S6).
S1) construction method of the sequencing library of the RNA in any of the above-described sample to be tested with poly (A) tail is dividing
Analyse the application in the sequence of the RNA in sample to be tested with poly (A) tail.
S2) construction method of the sequencing library of the RNA in any of the above-described sample to be tested with poly (A) tail is dividing
Analyse the application in poly (A) tail sequence of the RNA in sample to be tested with poly (A) tail.
S3) it is any of the above-described it is described it is unicellular in the construction method of sequencing library of the RNA with poly (A) tail analyzing
Application in the sequence of RNA in unicellular with poly (A) tail.
S4) it is any of the above-described it is described it is unicellular in the construction method of sequencing library of the RNA with poly (A) tail analyzing
Application in poly (A) tail sequence of RNA in unicellular with poly (A) tail.
S5) application of any of the above-described the method in the sequence that analysis has the RNA of poly (A) tail.
S6) any of the above-described the method answering in poly (A) tail sequence that analysis has the RNA of poly (A) tail
With.
The present invention also protects a kind of kit, it may include any of the above-described guidance primer, any of the above-described RT primer,
At least one of any of the above-described TSO and any of the above-described PCR primer.
The kit specifically can be by any of the above-described guidance primer, any of the above-described RT primer, any of the above-described institute
State TSO and any of the above-described PCR primer composition.
At least one of the present invention also protects the application of any of the above-described kit, can be T1)-T8):
T the sequence of the RNA in sample to be tested with poly (A) tail) is analyzed；
T2 poly (A) tail sequence of the RNA in sample to be tested with poly (A) tail) is analyzed；
T3 the sequence of the RNA in unicellular with poly (A) tail) is analyzed；
T4 poly (A) tail sequence of the RNA in unicellular with poly (A) tail) is analyzed；
T5) analysis has the sequence of the RNA of poly (A) tail；
T6) analysis has poly (A) tail sequence of the RNA of poly (A) tail；
T7 the sequencing library of the RNA in sample to be tested with poly (A) tail) is constructed；
T8 the sequencing library of the RNA in unicellular with poly (A) tail) is constructed.
Any of the above-described sample to be tested can be biological cell or biological tissue.
The total serum IgE of any of the above-described sample to be tested can be the total serum IgE of biological cell or the total serum IgE of biological tissue.It is described
Total serum IgE can be by purifying, can also be without purifying (containing a small amount of impurity, cell, tissue, extracting solution etc.).
It is any of the above-described it is described it is unicellular for biology it is unicellular.The biology is unicellular can be by purifying, can also not
By purifying (containing a small amount of impurity, separating liquid, mitochondria etc.).
Any of the above-described biology can be animal, plant or microorganism.The animal can be mouse, people, pig, ox, rabbit or sheep
Etc..The mouse can be mouse or rat.The mouse can be CD1 (ICR) mouse.The pig can be Large White.
Any of the above-described cell can be egg cell, cancer cell, leucocyte or plant (common) cell etc..The cancer is thin
Born of the same parents' concretely cervical cancer cell.The egg cell can be GV egg cell.
Any of the above-described tissue can be liver organization, renal tissue, spleen tissue or plant (common) tissue etc..
In an embodiment of the present invention, GV egg cell, the Hela of the sample to be tested concretely CD1 (ICR) mouse are thin
Born of the same parents or pig liver tissue.It is described unicellular for the single GV ovum of CD1 (ICR) mouse.
Present inventors have developed a kind of new sequencing approaches, are named as PAIso-seq (poly (A) inclusive
RNA isoform sequencing) method, accurately it can sensitively read different RNA hypotypes within the scope of transcript profile and be wrapped
Overall length poly (A) information contained.The step of PAIso-seq method, is as follows: connector is added first behind poly (A) tail
Retain poly (A) sequence；Then application template exchanges method to expand full-length cDNA, retains complete poly (A) tail；Finally
Implement three generations's sequencing using PacBio platform.The end extension mechanism of template dependant protects the integrality of poly (A) sequence, connects
Header sequence provides primer binding site for reverse transcription and PCR amplification.The end carried out by Oligo (dT) extends be template according to
Bad, it is only carried out in the RNA containing poly (A), avoids the enriching step of subsequent poly (A)+RNA.Template exchange
Using allowing the sensitivity of PAIso-seq method to compare favourably with most sensitive unicellular RNA-seq technology so far.
PacBio microarray dataset also has the advantage of accurate Analysis long-chain homopolymerization sequence.In addition, the ring-type that such technology platform uses is single
Molecule cycle sequencing strategy can implement multiple reading data to single cDNA molecule, to obtain the consistency base of high precision
Identify data.The present inventor using PAIso-seq method respectively mouse GV egg cell, the single GV ovum of mouse,
Poly (A) tail data in full transcript profile are analyzed in Hela cell and pig liver tissue on a large scale, these data include
The quantitative-length of poly (A) tail and accurate non-A modification analysis.The result shows that many mRNA in addition to 3 ' ends occur U and
Except G modification, the non-A modification of some U, G or C is also had occurred in the inside of poly (A) tail；The same gene it is transcribed go out not
With RNA hypotype, and poly possessed by the RNA of these specific subtypes (A) tail is different.While PAIso-seq method is also
Can Accurate Analysis poly (A) tail base composition.Analysis the result shows that, PAIso-seq data show many poly (A) tail
Inside, rather than the 3 ' end of tail thought in the past, also widely distributed non-A residue.Therefore, PAIso-seq method can be fine
Poly (A) tail of different RNA hypotypes is analyzed, the base composition of poly (A) tail is prompted to want more than what people before were thought
It is complicated.This also implies that the modification of poly (A) tail and regulation have the extremely complex and worth mechanism further excavated.These
As a result be not only that research poly (A) tail provides an accurate and sensitive new tool, also for the function of poly (A) tail and
Study on regulation opens new gate.PAIso-seq method is very sensitive, can be used for the RNA of single celled full transcript profile
Poly (A) analysis.
Detailed description of the invention
Fig. 1 is the flow diagram for constructing the PAIso-seq sequencing library of sample to be tested.
Fig. 2 is Dnmt1 gene, Cnot7 gene, Btg4 gene and Plat gene in analysis GV egg cell and single GV ovum
Poly (A) tail length.
Fig. 3 is poly (A) information analyzed in transcript.GV rep.1 is that GV egg cell sample repetition 1, GV rep.2 is
GV egg cell sample repeats the mixing sample that 2, SCGV com. is single GV ovum, and blue line is linear regression line, light gray areas
It indicates to return confidence interval, the transcript quantity with corresponding length poly (A) tail has been marked in figure.
Fig. 4 is the poly of all transcripts in the mixing sample data of single GV ovum, GV egg cell and single GV ovum
(A) tail length distribution, non-A modification frequency and spearman correlation analysis.GV rep.2 is that GV egg cell sample repeats 2,
SCGV com. is the mixing sample of single GV ovum, and blue line is linear regression line, and light gray zones domain representation returns confidence interval.
Fig. 5 is that poly (A) the tail length distribution of transcript in Hela cell and non-A modify frequency.
Fig. 6 is that poly (A) the tail length distribution of transcript in pig liver tissue and non-A modify frequency.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
Test material as used in the following examples is unless otherwise specified to buy from routine biochemistry reagent shop
It arrives.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
Embodiment 1, the PAIso-seq sequencing library for constructing sample to be tested
Due to that can not parse long-chain homopolymerization sequence, current Illumina platform cannot provide for analysis poly (A) tail
One good tool.TAIL-seq and PAL-seq technology is special using special poly (T) length analysis algorithm or application
The available poly of sequencing approach (T) length information, but both methods can not identify be distributed in poly (A) 3 ' end
Non- A modification except end regions.In addition, TAIL-seq and PAL-seq method is required to the RNA sample of milligram grade, this defect
Limit their applications in precious biopsy samples and Patient Sample A.Recently the PacBio three generations's sequencing technologies developed, pass through
Real time sequence reading is carried out to individual molecule, can identify the base composition of parsing long-chain homopolymerization sequence.In addition, being cyclized in library
The application of sequencing template is read several times single template, obtains CCS sequence (Circular using the method
Consensus Sequence) information, greatly improve the accuracy of sequence analysis.Thus, PacBio three generations's microarray dataset
It may be the optimal selection for parsing poly (A) tail length and base composition in RNA.
The present inventor passes through many experiments, and the PAIso-seq sequencing library by constructing sample to be tested accurately divides
Analyse the full-length RNA sequence and its poly (A) tail sequence of several target gene in sample to be tested.Specific step is as follows:
1, end extends
The total serum IgE of sample to be tested is taken, is added dNTP and Klenow segment (exo-, NEB), is mixed；Then with guidance primer
Carry out end extension.
Guide primer from 5 ' ends to 3 ' ends successively include DNA fragmentation first, DNA fragmentation second and DNA fragmentation third；DNA piece
Duan Jiawei arbitrary sequence (length 15-40bp) is used for PCR amplification；DNA fragmentation second is Barcode sequence (length 8-
30bp), for distinguishing different PAIso-seq sequencing libraries；DNA fragmentation third is made of 3-24bp nucleotide, each nucleotide
For uracil-DNA nucleotide (being indicated with dU) or T；At least one in 3 nucleotide of DNA fragmentation the third 5 ' end is dU.
End extension condition are as follows: 37 DEG C of incubation 1h.
2, USER digestion and RNA purifying
(1) USER enzyme (NEB) is added into the reaction system for completing step 1,37 DEG C of incubation 1h.
USER enzyme can identify and cut the dU residue in guidance primer, and then cut off guidance primer, avoid guidance primer
Primer as reverse transcription.
(2) nucleic acid fragment that recycling length is 200nt or more.
The method of recycling specifically: 50 μ L RNA Binding buffer are added into the reaction system for completing step (1)
(component in RNA Clean&Concentrator-5 kit) and 50 μ L ethyl alcohol mix, obtain mixed solution；It will mix molten
Liquid is transferred to Zymo-Spin IC pillar, centrifugation；Utilize RNAClean&Concentrator-5 kit (Zymo
Research pillar, centrifugation) are cleaned；6-8 μ L, which is added, to base for post goes nuclease water to be eluted, and is centrifuged, obtains nucleic acid solution；It should
Nucleic acid fragment in nucleic acid solution extends by end and length is 200nt or more.
3, C is shifted in reverse transcription and end
The nucleic acid solution that step 2 obtains is taken, carries out reverse transcription with RT primer, since reverse transcriptase is living with terminal enzyme (DNA)
Property, so reverse transcriptase can add C in 5 ' ends of reverse transcription product, obtain the cDNA that 5 ' ends have C flag.
RT primer is all or part of of the nucleotide sequence of DNA fragmentation first.
4, template replacement
The reaction system for taking into step 3 carries out template exchange with TSO, obtains cDNA.
TSO from 5 ' ends to 3 ' ends successively include DNA fragmentation fourth and nucleic acid fragment fourth；DNA fragmentation fourth is arbitrary sequence
(length 15-40bp)；Nucleic acid fragment fourth is made of section 1 and section 2；Section 1 is any DNA sequence (length 0-
10bp)；Section 2 is by N number of rG or " (N-1) a rG and 1 G*" composition, the natural number that N is 3 or more；RG indicates guanine ribose
Nucleotide, G*It indicates to have carried out the guanine deoxyribonucleotide that lock nucleic acid is modified.
DNA fragmentation Ding Kewei DNA fragmentation first.
5, the amplification of cDNA
The cDNA obtained using step 4 carries out PCR amplification as template, using upstream primer and downstream primer, is expanded
cDNA。
Upstream primer is all or part of of the nucleotide sequence of DNA fragmentation fourth.
Downstream primer is all or part of of the nucleotide sequence of DNA fragmentation first.
If DNA fragmentation fourth and DNA fragmentation first include DNA fragmentation oneself, upstream primer and downstream primer be DNA fragmentation oneself
Nucleotide sequence it is all or part of, upstream primer and downstream primer can be identical at this time, i.e., carry out PCR using single primer
Amplification.
6, it is cyclized the connection of connector
The amplification cDNA obtained using Pure PB beads to step 5 carries out length selection, specially with 1 × beads
Selection is greater than the amplification cDNA of 200bp, with 0.4 × beads selection be greater than the amplification cDNA of 2kb.It will be greater than the amplification of 200bp
CDNA and greater than 2kb amplification cDNA equimolar mixing, then use SMRTbell Template Prep kit
(PacBio) library SMRTbell Template is constructed.
7, PacBio is sequenced
The library SMRTbell Template is sequenced using PacBio platform, obtains the PAIso-seq of sample to be tested
Sequencing library.
It, can be with the full-length RNA sequence of target gene in Accurate Analysis sample to be tested and its poly (A) tail according to sequencing result
Ba Xulie.
The flow diagram of the PAIso-seq sequencing library of sample to be tested is shown in Fig. 1 (DNA fragmentation fourth and DNA fragmentation first phase
Together).
It should be noted that if sample to be tested be it is unicellular, " total serum IgE for taking sample to be tested " in step 1 is replaced
" to take unicellular, aqueous solution and 1 μ L RNase inhibitor of the 19 μ L of addition containing 0.2% (v/v) TritonX-100,80 DEG C of incubations
5min, cracking, obtains cell pyrolysis liquid ", other steps are constant, obtain single celled PAIso-seq sequencing library.
Embodiment 2, embodiment 1 construct sample to be tested PAIso-seq sequencing library analysis mouse GV egg cell and
The application of transcript poly (A) tail length in the single GV ovum of mouse
1, it is injected intraperitoneally to CD1 (ICR) mouse (Beijing Vital River Experimental Animals Technology Co., Ltd.) of 7-8 week old pregnant
Horse serum promoting sexual gland hormone (ProSpec) then obtains ovum using oothecocentesis (No. 30 needles)；Ovum is first cultivated with M2
Base (Sigma) is washed 1 time, then is washed 3 times with PBS (PBSA) buffer containing 0.1% (v/v) BSA, and GV egg cell is obtained.
2, GV egg cell is taken, is extracted using Direct-zol RNA MicroPrep kit (Zymo Research) total
RNA obtains the total serum IgE of GV egg cell.Specific step is as follows:
(1) GV egg cell is taken, 500 μ L TRIzol (Ambion) cracking is added, thoroughly mixes；
(2) 500 μ L ethyl alcohol are added into the system for completing step (1), thoroughly mix；
(3) system for completing step (2) is transferred to Zymo-Spin IC pillar, be centrifuged；Then pillar is cleaned, is centrifuged；
(4) after completing step (3), 10-30 μ L is added to base for post, nuclease water is gone to be eluted, be centrifuged, it is thin to obtain GV ovum
The total serum IgE of born of the same parents.
3, " total serum IgE of sample to be tested " in embodiment 1 is replaced with to the total serum IgE of GV egg cell, other steps are constant, obtain
To the PAIso-seq sequencing library of GV egg cell.The experiment is repeated twice.
4, " total serum IgE for taking sample to be tested " in 1 step 1 of embodiment is replaced with and " takes single GV ovum, 19 μ L are added and contain
The aqueous solution of 0.2% (v/v) TritonX-100 and 1 μ L RNase inhibitor, 80 DEG C of incubation 5min, cracking obtain cell cracking
Liquid ", other steps is constant in step 1 and 2, obtains single celled nucleic acid solution；By 15 single celled nucleic acid solutions (by
15 single GV ovums obtain respectively) mixing, it obtains mixing nucleic acid solution and (mixes in nucleic acid solution, each single celled nucleic acid
It is identical in quality)；" nucleic acid solution " in 1 step 3 of embodiment is replaced with into mixing nucleic acid solution, step 3 to 7 is constant, obtains list
The PAIso-seq sequencing library of the mixing sample of a GV ovum.The experiment is repeated once.
5, " total serum IgE for taking sample to be tested " in 1 step 1 of embodiment is replaced with and " takes single GV ovum, 19 μ L are added and contain
The aqueous solution of 0.2% (v/v) TritonX-100 and 1 μ L RNase inhibitor, 80 DEG C of incubation 5min, cracking obtain cell cracking
Liquid ", other steps are constant, obtain the PAIso-seq sequencing library of single GV ovum.15 GV ovums are taken (to order respectively altogether
Entitled C1-C15), correspondingly, obtaining the PAIso-seq sequencing library of 15 GV ovums.
In step 3, step 4 and step 5, the nucleotide sequence of each primer is as follows: guidance primer: (box is Barcode sequence)；RT primer: 5 '-AAGCAGTGGTATCAACGCAGAGTAC-3 '；TSO:
5’-AAGCAGTGGTATCAACGCAGAGTACATrGrGG*-3'.Since DNA fragmentation fourth is identical as DNA fragmentation first, then carry out
The upstream primer and downstream primer of PCR amplification are also identical, i.e., carry out PCR amplification using single primer；Carry out the primer of PCR amplification:
5’-AAGCAGTGGTATCAACGCAGAGT-3’。
6, according to the sequencing result of step 3 and step 4, in the mixing sample for analyzing GV egg cell and single GV ovum
Dnmt1 gene, Cnot7 gene, Btg4 gene and Plat gene poly (A) tail length, poly (A) tail length is with middle position
Number indicates；And analyze poly (A) information captured in transcript.
The analysis result of poly (A) tail length is shown in that (GV rep.1 is that GV egg cell sample repeats 1, GV to left figure in Fig. 2
Rep.2 is the mixing sample that GV egg cell sample repeats that 2, SCGV com. is single GV ovum, is followed successively by Dnmt1 from top to down
Gene, Cnot7 gene, Btg4 gene and Plat gene).The result shows that poly (A) tail of DNMT1 gene is longer, and
Poly (A) tail of Cnot7 gene, Btg4 gene and Plat gene is shorter.Transcript number has between 3 repetitions
Spearman correlation (A in Fig. 3).
Poly (A) tail length has spearman correlation (B in Fig. 3) between 3 repetitions.
7, according to the sequencing result of step 3, step 4 and step 5, single GV ovum, GV egg cell and single GV ovum are analyzed
Poly (A) the tail length distribution of all transcripts and non-A modify frequency in the mixing sample data of son, analyze C15 and C4, C4
With GV rep.2, the spearman correlation of poly (A) tail length between C15 and GV rep.2.
Poly (A) tail length Distributed parts result of all transcripts is shown in that (red dot and number below are A in Fig. 4
Poly (A) tail length median).
Non- A modification frequency-portions result is shown in B in Fig. 4.
Poly (A) tail length has spearman correlation (the upper figure of C in Fig. 4) between C15 and C4.
Poly (A) tail length has spearman correlation (the middle figure of C in Fig. 4) between C4 and GV rep.2.
Poly (A) tail length has spearman correlation (following figure of C in Fig. 4) between C15 and GV rep.2.
8, using PAT joint Fragment Analyzer Capillary Electrophoresis method (be recorded in following document:
Mishima, Yuichiro, Tomari, Yukihide.Codon Usage and3 ' UTR Length Determine
Maternal mRNA Stability in Zebrafish.10.1016/j.molcel.2016.02.027) the single GV of measurement
Poly (A) tail length of Dnmt1 gene, Cnot7 gene, Btg4 gene and Plat gene in ovum.Experiment takes in triplicate
Average value.
Testing result is shown in right figure in Fig. 2.The result shows that poly (A) tail of DNMT1 gene is longer, and Cnot7 gene,
Poly (A) tail of Btg4 gene and Plat gene is shorter, this is completely the same with the result of left figure in Fig. 2.
The above results show that the PAIso-seq sequencing library of GV egg cell can analyze transcript in GV egg cell
The PAIso- of the mixing sample of poly (A) tail length, the PAIso-seq sequencing library of single GV ovum or single GV ovum
Seq sequencing library can analyze poly (A) tail length of transcript in single GV ovum.
The PAIso-seq sequencing library for the sample to be tested that embodiment 3, embodiment 1 construct is transcribed in analysis Hela cell
The application of this poly (A) tail length and non-A modification frequency
1, Hela cell (ATCC) is taken, total serum IgE is extracted using Direct-zol RNA MicroPrep kit, is obtained
The total serum IgE of Hela cell.Specific step is as follows:
(1) Hela cell is taken, 500 μ L TRIzol cracking is added, thoroughly mixes；
(2) 500 μ L ethyl alcohol are added into the system for completing step (1), thoroughly mix；
(3) system for completing step (2) is transferred to Zymo-Spin IC pillar, be centrifuged；Then pillar is cleaned, is centrifuged；
(4) after completing step (3), 10-30 μ L is added to base for post, nuclease water is gone to be eluted, be centrifuged, it is thin to obtain Hela
The total serum IgE of born of the same parents.
2, " total serum IgE of sample to be tested " in embodiment 1 is replaced with to the total serum IgE of Hela cell, other steps are constant, obtain
To the PAIso-seq sequencing library of Hela cell.The experiment is repeated twice.
Guide primer, RT primer, TSO, carry out PCR amplification primer nucleotide sequence with corresponding sequence in embodiment 2
Column.
3, according to the sequencing result of step 2, the distribution of transcript poly (A) tail length and non-A in Hela cell is analyzed and is repaired
Adorn frequency.
The result that transcript poly (A) tail length is distributed in Hela cell is shown in left figure in Fig. 5.
Right figure in the result figure 5 of non-A modification frequency in Hela cell.
The result shows that the PAIso-seq sequencing library of Hela cell can analyze the poly (A) of transcript in Hela cell
Tail length and non-A modify frequency.
The PAIso-seq sequencing library for the sample to be tested that embodiment 4, embodiment 1 construct is in analysis pig liver tissue transfer
Record the application of poly (A) tail length originally and non-A modification frequency
1, pig liver tissue (kind of pig is Large White) is taken, is mentioned using Direct-zol RNAMicroPrep kit
Total serum IgE is taken, the total serum IgE of pig liver tissue is obtained.Specific step is as follows:
(1) pig liver tissue is taken, 500 μ L TRIzol cracking is added, thoroughly mixes；
(2) 500 μ L ethyl alcohol are added into the system for completing step (1), thoroughly mix；
(3) system for completing step (2) is transferred to Zymo-Spin IC pillar, be centrifuged；Then pillar is cleaned, is centrifuged；
(4) after completing step (3), 10-30 μ L is added to base for post, nuclease water is gone to be eluted, be centrifuged, obtain pig liver
The total serum IgE of tissue.
2, " total serum IgE of sample to be tested " in embodiment 1 is replaced with to the total serum IgE of pig liver tissue, other steps are constant,
Obtain the PAIso-seq sequencing library of pig liver tissue.The experiment is repeated twice.
Guide primer, RT primer, TSO, carry out PCR amplification primer nucleotide sequence with corresponding sequence in embodiment 2
Column.
3, according to the sequencing result of step 2, the distribution of transcript poly (A) tail length and non-A in pig liver tissue are analyzed
Modify frequency.
The result that transcript poly (A) tail length is distributed in pig liver tissue is shown in left figure in Fig. 6.
Right figure in the result figure 6 of non-A modification frequency in pig liver tissue.
The result shows that the PAIso-seq sequencing library of pig liver tissue can analyze transcript in pig liver tissue
Poly (A) tail length and non-A modify frequency.
The length of PAIso-seq sequencing library detection poly (A) tail for the sample to be tested that embodiment 5, embodiment 1 construct
Accuracy
1, it is synthesized by Takara company referring to primer spike-in, and is gone out before initiation codon with barcode label
(spike-in tail length is respectively set to 10,30,50,70 and 100 to poly (A) tail of different length in spike-in
A)。
2, after completing step 1, respectively with the mcherry carrier of the spike-in of poly (A) tail containing different length
PCR amplification is carried out for template, obtains the double-stranded DNA of different length spike-in, then carries out PCR amplification production according to different length
The recycling of object glue.
3, pcr amplification product is taken, SMRTbell Template is constructed using SMRTbell Template Prep kit
Library.
4, the library SMRTbell Template is sequenced using PacBio platform.
According to sequencing result, the PAIso-seq of Spike-in is analyzed in data, poly (A) tail length median difference
It is 10,28,48,67 and 97.It can be seen that the PAIso-seq sequencing library for the sample to be tested that embodiment 1 constructs can be to poly
(A) length of tail carries out accurate quantitative description.
The above results show the PAIso-seq sequencing library energy accurate response poly for the sample to be tested that embodiment 1 constructs
(A) length of tail.
Claims (10)
1. the construction method of the sequencing library of the RNA in sample to be tested with poly (A) tail, in turn includes the following steps:
(a1) total serum IgE for taking sample to be tested carries out end extension using guidance primer, USER enzyme is then added and sufficiently digests, returns
Receive the nucleic acid fragment that length is 200nt or more；
Guide primer from 5 ' ends to 3 ' ends successively include DNA fragmentation first and DNA fragmentation third；
DNA fragmentation first is the arbitrary sequence of 15-40bp and cannot be used for PCR amplification in conjunction with poly (A) tail；
DNA fragmentation third is made of 3-24bp nucleotide, and each nucleotide is dU or T；3 nucleotide of DNA fragmentation the third 5 ' end
In at least one be dU；
(a2) nucleic acid fragment is taken, reverse transcription is carried out with RT primer, obtains cDNA；
RT primer is all or part of of the nucleotide sequence of DNA fragmentation first；
(a3) cDNA is taken, carries out template exchange with TSO；
TSO from 5 ' ends to 3 ' ends successively include DNA fragmentation fourth and nucleic acid fragment fourth；
DNA fragmentation fourth is the arbitrary sequence of 15-40bp, is used for PCR amplification；
Nucleic acid fragment fourth includes section 2, and section 2 is used for and the cDNA is combined；Section 2 by N number of guanosine ribonucleoside acid or
" the guanine deoxyribonucleotide that (N-1) a guanosine ribonucleoside acid and 1 have carried out lock nucleic acid modification " composition, N 3
Above natural number；
(a4) cDNA for taking into step (a3) carries out PCR amplification using PCR primer, obtains amplification cDNA；
Upstream primer in PCR primer is all or part of of the nucleotide sequence of DNA fragmentation fourth；
Downstream primer in PCR primer is all or part of of the nucleotide sequence of DNA fragmentation first；
(a5) the amplification cDNA is taken, is sequenced；Obtain the sequencing library of the RNA in sample to be tested with poly (A) tail.
2. the construction method of the sequencing library of the RNA in unicellular with poly (A) tail, in turn includes the following steps:
(b1) it is cracked unicellular, obtains cell pyrolysis liquid；Then end extension is carried out using guidance primer；It is added later
USER enzyme sufficiently digests, the nucleic acid fragment that recycling length is 200nt or more；
Guide primer from 5 ' ends to 3 ' ends successively include DNA fragmentation first and DNA fragmentation third；
DNA fragmentation first is the arbitrary sequence of 15-40bp and cannot be used for PCR amplification in conjunction with poly (A) tail；
DNA fragmentation third is made of 3-24bp nucleotide, and each nucleotide is dU or T；3 nucleotide of DNA fragmentation the third 5 ' end
In at least one be dU；
(b2) nucleic acid fragment is taken, reverse transcription is carried out with RT primer, obtains cDNA；
RT primer is all or part of of the nucleotide sequence of DNA fragmentation first；
(b3) cDNA is taken, carries out template exchange with TSO；
TSO from 5 ' ends to 3 ' ends successively include DNA fragmentation fourth and nucleic acid fragment fourth；
DNA fragmentation fourth is the arbitrary sequence of 15-40bp, is used for PCR amplification；
Nucleic acid fragment fourth includes section 2, and section 2 is used for and the cDNA is combined；Section 2 by N number of guanosine ribonucleoside acid or
" the guanine deoxyribonucleotide that (N-1) a guanosine ribonucleoside acid and 1 have carried out lock nucleic acid modification " composition, N 3
Above natural number；
(b4) cDNA for taking into step (b3) carries out PCR amplification using PCR primer, obtains amplification cDNA；
Upstream primer in PCR primer is all or part of of the nucleotide sequence of DNA fragmentation fourth；
Downstream primer in PCR primer is all or part of of the nucleotide sequence of DNA fragmentation first；
(b5) the amplification cDNA is taken, is sequenced；Obtain the sequencing library of the RNA in unicellular with poly (A) tail.
3. method according to claim 2, it is characterised in that: in the step (b1), " crack, obtain by unicellular
The method of cell pyrolysis liquid " are as follows: to unicellular middle addition nonionic surface active agent and RNase inhibitor, be incubated for, obtain thin
Cellular lysate liquid.
4. the method as described in claims 1 to 3 is any, it is characterised in that: described to draw in the step (a1) and step (b1)
Guiding object further includes DNA fragmentation second, and DNA fragmentation second is located at the 5 ' ends at 3 ' ends of DNA fragmentation first, DNA fragmentation third；DNA fragmentation second
For the Barcode sequence of 8-30bp, for distinguishing different sample to be tested or unicellular.
5. the method as described in Claims 1-4 is any, it is characterised in that: in the step (a3) and step (b3), the core
Acid fragment fourth includes section 1, and section 1 is located at 5 ' ends of section 2；The section 1 is the arbitrary sequence of 0-10bp.
6. method as claimed in claim 1 to 5, it is characterised in that: described in the step (a3) and step (b3)
DNA fragmentation fourth is DNA fragmentation first.
7. the method as described in claim 1 to 6 is any, it is characterised in that: in the step (a5) and step (b5), " take expansion
Increase cDNA, sequencing " are as follows: it will be greater than the amplification cDNA of 200bp and the amplification cDNA mixing greater than 2kb, use SMRTbell later
Template Prep kit constructs the library SMRTbell Template；Using PacBio platform to SMRTbell
The library Template is sequenced.
Any one of 8.S1)-S6):
S1) the sequence of the RNA with poly (A) tail in analysis sample to be tested of method described in claim 1,4,5,6 or 7
In application；
S2) the poly of the RNA with poly (A) tail in analysis sample to be tested of method described in claim 1,4,5,6 or 7
(A) application in tail sequence；
S3) any method of claim 2 to 7 is in the sequence for analyzing the RNA in unicellular with poly (A) tail
Using；
S4) any method of claim 2 to 7 is in the poly (A) for analyzing the RNA in unicellular with poly (A) tail
Application in tail sequence；
S5) application of any method of claim 1 to 7 in the sequence that analysis has the RNA of poly (A) tail；
S6) any method of claim 1 to 7 has poly (A) tail sequence of the RNA of poly (A) tail in analysis
In application.
9. a kind of kit, including at least one of primer, RT primer, TSO and PCR primer are guided in claim 1 to 6.
At least one of 10. the application of kit described in claim 9 is T1)-T8):
T the sequence of the RNA in sample to be tested with poly (A) tail) is analyzed；
T2 poly (A) tail sequence of the RNA in sample to be tested with poly (A) tail) is analyzed；
T3 the sequence of the RNA in unicellular with poly (A) tail) is analyzed；
T4 poly (A) tail sequence of the RNA in unicellular with poly (A) tail) is analyzed；
T5) analysis has the sequence of the RNA of poly (A) tail；
T6) analysis has poly (A) tail sequence of the RNA of poly (A) tail；
T7 the sequencing library of the RNA in sample to be tested with poly (A) tail) is constructed；
T8 the sequencing library of the RNA in unicellular with poly (A) tail) is constructed.
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