CN106801050A - A kind of construction method and its kit of circular rna high-throughput sequencing library - Google Patents
A kind of construction method and its kit of circular rna high-throughput sequencing library Download PDFInfo
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Abstract
The invention discloses a kind of construction method and its kit of circular rna high-throughput sequencing library, the construction method is comprised the following steps successively：(S1) total serum IgE in sample is extracted；(S2) DNA in removal sample；(S3) detect and evaluate the total serum IgE quality in sample, determine RNA satisfactory qualities；(S4) rRNA is removed；(S5) linear rna is removed；(S6) the circular rna library for high-flux sequence is built.The construction method of circular rna high-throughput sequencing library of the present invention efficiently, stabilization, rRNA residual ratios are low, and circular rna detector efficiency is high, and preferably, it is high that sequencing result is proved to be successful rate to data redundancy, is particularly suited for the ropy samples of RNA such as FFPE tissues.
Description
Technical field
The invention belongs to biology field, and in particular to a kind of construction method of circular rna high-throughput sequencing library
And its kit.
Background technology
Circular rna (ciucular RNA, circRNA) is a kind of RNA families newcomer for being different from conventional linear RNA,
It is the non-coding RNA molecule that loop configuration is formed without 5 ' end caps and 3 ' end poly (A) tails and with covalent bond.
Early in 1980, circular rna just advantageously, it has been found that still within a period of time very long, due to the limitation of investigative technique level,
The byproduct that circular rna is considered as wrong variable sheer and is formed, belongs to a kind of extremely rare phenomenon in nature, and even
It is taken as caused by hereditary unexpected or experiment human factor, does not cause academic attention.With depth RNA sequencings and scale metaplasia
The development of thing information technology, researcher just has found there are a large amount of circular RNA molecules in vivo.Salzman in 2012 et al.
It is found that circular rna is widely present in the middle of the transcript profile of people by the method for high-flux sequence, so as to cause people to circular rna
The interest of research.Hansen in 2013 et al. has found that CDR1as/CiRS-7 can be expressed as miR-7 sponge controlling genes,
Confirm important function of the circular rna in human body.Recent studies have shown that circular rna has closed hoop structure, mainly pass through
The processing of atypia variable sheer is produced, and is widely present in various biological cells, with Stability Analysis of Structures, it is difficult to degraded by RNase,
Gene expression abundance is high, conservative is good between species, and expression has the features such as tissue and Space-time speciality.Display ring-type is studied at present
RNA is related with diseases such as neurodevelopment, atherosclerosis, myotonia dystrophy, cancers, and in people's saliva and blood
In detect the presence of circular rna, then show that circular rna can be in blood, urine, stable existence in the clinical samples such as ascites,
These features cause that circular rna has broad prospects in the development and application of New Type of Diseases Clinics and Practices method.
The A of CN 104388548 disclose a kind of method of high flux circular rna sequencing, and it includes：Artificial synthesized external source line
Property ERCC-0004-RNA and external source ring-type ERCC-0013-RNA；Quality control is carried out to external source ring-type ERCC-0013-RNA；Will
The linear ERCC-0004-RNA of external source and external source ring-type ERCC-0013-RNA, than mixing, is obtained mixing exogenous RNA by equimolar；To
Mixing exogenous RNA is added in the total serum IgE of sample to be sequenced, the first mixture is obtained；Remove the ribosomes in the first mixture
RNA, obtains the second mixture；3 ' terminal biotins mark is carried out to the second mixture, and removes the lasso trick of the upper biotin of mark
RNA and linear rna, obtain the 3rd mixture；The linear rna in the 3rd mixture is removed, the 4th mixture is obtained；It is mixed to the 4th
Compound carries out the high flux transcript profile sequencing of standard and carries out biological analysis to sequencing data.The method step is complicated, and needs
Want pre-synthesis exogenous RNA.
Because circular rna cannot be directly separated, so do not have a mark for circular rna library construction in document so far
Accurate method, using removing rRNA and removing linear rna in most of researchs, then builds for high-throughput sequencing library again
Method, it is directly to build storehouse after removal rRNA also to have sub-fraction research, or directly builds storehouse after removing linear rna, also minimum
Part research is the method using storehouse is built after removal PolyA+RNA.There is rRNA removals not thoroughly in these methods, Data duplication
Property difference the shortcomings of.Therefore, the library for circular rna high-flux sequence how efficiently, is stably built, is still urgently to be resolved hurrily
A technical barrier.
Additionally, also build storehouse kit without special circular rna in the market, it is necessary to scientific research personnel from QIAGEN,
Illumina, Backman, NEB, Life technology Deng Duo companies buy respectively RNA extract, it is rRNA removals, linear
The products such as RNA removals, library construction, then groping suitable condition carries out building storehouse.On the one hand this many reagent sets build storehouse jointly
Expensive, the scientific research personnel on the other hand needing experimental skill higher spends longer time to go to grope most preferably to test bar
Part, seriously limits circular rna progress of research, it is therefore necessary to develops a energy and efficiently, stably builds for circular rna
The dedicated kit of high-throughput sequencing library.
The content of the invention
In view of the above problems, inventor carries out research and analysis repeatedly to circular rna sequencing, develops a kind of energy
Enough methods for efficiently and stably building circular rna high-throughput sequencing library, the method rRNA residual ratios are low, data redundancy compared with
It is good, and the method is particularly suited for the ropy samples of RNA such as FFPE tissues.
To achieve these goals, the present invention provides a kind of construction method of circular rna high-throughput sequencing library, and it is successively
Comprise the following steps：
(S1) total serum IgE in sample is extracted；
(S2) DNA in removal sample；
(S3) detect and evaluate the total serum IgE quality in sample, determine RNA satisfactory qualities；
(S4) rRNA is removed；
(S5) linear rna is removed；
(S6) the circular rna library for high-flux sequence is built.
Preferably, in the construction method of the circular rna high-throughput sequencing library, sample is extracted in the step (S1)
In total serum IgE carried out using Trizol methods, the method is particularly well-suited to tissue and cell sample.
Preferably, in the construction method of the circular rna high-throughput sequencing library, removal sample in the step (S2)
In DNA carried out using DNase I digestion methods.
Preferably, in the construction method of the circular rna high-throughput sequencing library, detection sample in the step (S3)
In total RNA mass comprise the following steps successively：
(S31) RNA palliating degradation degrees are detected using 1% agarose gel electrophoresis and whether has pollution；
(S32) using the purity of UV spectrophotometer measuring RNA, i.e. OD260/280 ratios；
(S33) integrality of RNA is detected.
It is highly preferred that in the construction method of the circular rna high-throughput sequencing library, detection in the step (S33)
The integrality of RNA is carried out using the biological analysers of Agilent 2100.
In the present invention, OD (optical density) represents the optical density that tested substance is sponged, OD260/280
Ratio refers to the absorption photometric ratio at wavelength 260nm and 280nm, the purity for judging RNA.In theory, in pure rna
In the case of OD260/280 ratios be 2, wherein OD260 represents the absorbance of nucleic acid, and OD280 represents the absorbance of protein.
Preferably, in the construction method of the circular rna high-throughput sequencing library, removal rRNA in the step (S4)
Carried out using RNase H digestion methods.
Preferably, in the construction method of the circular rna high-throughput sequencing library, the step (S4) removal rRNA according to
It is secondary to comprise the following steps：
(S41) by mass mixings such as the DNA probes complementary with rRNA sequences, DNA probe storehouse is formed；
(S42) obtained DNA probe storehouse is detected and evaluates the total serum IgE quality in sample with process and determine that RNA mass is accorded with
Desired RNA bulk crossings are closed, DNA-RNA hybrids are formed；
(S43) obtained DNA-RNA hybrids are digested with RNase H；
(S44) DNA probe is digested with DNase I, obtains the RNA that rRNA exhausts；
(S45) concentration of the RNA that rRNA exhausts is determined by fluorescence photometer.
Preferably, it is used in the step (S42) in the construction method of the circular rna high-throughput sequencing library
DNA probe storehouse is (1-2) with the weight ratio of RNA：1, preferably 1:1.
Preferably, in the construction method of the circular rna high-throughput sequencing library, mixing is miscellaneous in the step (S42)
It is to hybridize 2 minutes at a temperature of 95 DEG C to hand over used condition, then with the speed of 0.1 DEG C/s by temperature be slowly decreased to
45℃。
Preferably, in the construction method of the circular rna high-throughput sequencing library, RNase is used in the step (S43)
The condition that the obtained DNA-RNA hybrids of H digestion are used is to be digested 30 minutes at a temperature of 37 DEG C.
Preferably, in the construction method of the circular rna high-throughput sequencing library, determined in the step (S45)
The fluorescence photometer that the concentration of the RNA that rRNA exhausts is used is carried out by Qubit fluorescence photometers.
Preferably, in the construction method of the circular rna high-throughput sequencing library, removal is linear in the step (S5)
RNA is carried out using RNase R digestion methods, and the amount ratio of wherein RNase R and RNA is (2.5-3.5) U:1ug, preferably 3:
1, i.e., 3 RNase R of active unit (U) are added in every 1 microgram RNA.
It is highly preferred that in the construction method of the circular rna high-throughput sequencing library, removal line in the step (S5)
Property the digestion conditions of RNase R digestion methods that are used of RNA at 35-45 DEG C, 20-40 points to be digested at a temperature of preferably 37 DEG C
Clock, preferably 30 minutes.
Preferably, in the construction method of the circular rna high-throughput sequencing library, being built in the step (S6) is used for
The circular rna library of high-flux sequence is carried out using dUTP methods.
Preferably, in the construction method of the circular rna high-throughput sequencing library, being built in the step (S6) is used for
The circular rna library of high-flux sequence comprises the following steps successively：
(S61) RNA fragmentations；
(S62) the first chain synthesis：Carried out using the method for reverse transcriptase plus random primer；
(S63) the second chain synthesis：It is then poly- by DNA using the RNA chains in RNase H digestion RNA/cDNA hybrids
Synthase I synthesizes Article 2 chain；
(S64) end is repaired：The dsDNA of flat end is formed using End Repair Mix and at 5' ends plus phosphate
Group；
(S65) A tails are added：Add A tails in dsDNA ends using Klenow 3'-5'exo-；
(S66) Y types Adapter connections：Using the T4 DNA Ligase connection Adapter and dsDNA for having added A tails；
(S67) digestion of the second chain and amplified library：Using the chain of UNG enzymic digestions second；
(S68) quality inspection and preparation index library.
Preferably, in the construction method of the circular rna high-throughput sequencing library, RNA fragments in the step (S61)
The condition of change be at 90-96 DEG C, fragmentation 3-6 minutes at a temperature of preferably 94 DEG C, preferably 5 minutes.
Preferably, in the construction method of the circular rna high-throughput sequencing library, RNA fragments in the step (S61)
Peak value after change is located at 300-350bp.
Preferably, in the construction method of the circular rna high-throughput sequencing library, the chain of step (S62) first is closed
Into the DNA polymerase activity of the dependence DNA for suppressing reverse transcriptase using actinomycin D, to retain the directivity information of chain.
Preferably, in the construction method of the circular rna high-throughput sequencing library, step (S66) the Y types
Adapter ends connect T using phosphorothioate bond in Adapter connections, to form Adapter dimers to prevent, further
Preferably, after connection product purifying is using 1 × beads purifying once, 0.7 ×/0.2 × beads carries out clip size screening, with
Select the fragment of suitable size.
Preferably, in the construction method of the circular rna high-throughput sequencing library, step (S68) quality inspection and standard
Standby index library comprises the following steps successively：
(S681) library concentration is detected；
(S682) Library Quality is detected using Agilent 2100；
(S683) according to library concentration, different index libraries is mixed, machine sequencing in preparation.
Compared with conventional method, the construction method of circular rna high-throughput sequencing library provided by the present invention is efficiently, surely
Fixed, rRNA residual ratios are low, and circular rna detector efficiency is high, and preferably, it is high that sequencing result is proved to be successful rate to data redundancy, especially
Suitable for ropy samples of RNA such as FFPE tissues.
Additionally, the present invention also provides the kit that a kind of circular rna high-throughput sequencing library builds, it includes：
(1) circular rna enrichment reagents, DNase I, RNase H, RNaseR；
(2) the special DNA probe of kind；
(3) the various enzymes needed for library construction：DNA polymerase i, Large (Klenow) Fragment, T4
Polynucleotide Kinase(T4 PNK)、Klenow Fragment、Escherichia coli UDG、T4 DNA
Ligase(Rapid)、Phusion High-Fidelity DNA Polymerase；
(4) magnetic bead, dNTP, Nuclease Free Water, dUTP, joint, primer etc.,
Wherein, the special DNA probe of the kind can be complementary with the rRNA sequences of corresponding species.
Circular rna high-throughput sequencing library structure kit provided by the present invention is simple to operate, condition optimizing, cost
It is low, it is a powerful of circular rna research.
Compared with prior art, the present invention has advantages below and beneficial effect：
(1) construction method of circular rna high-throughput sequencing library of the present invention is because establish the special DNA probe of kind
Storehouse, the rRNA combined with probe using RNaseH removals, and circular rna is further enriched with using RNaseR, so rRNA is remained
Ratio is low, and circular rna detector efficiency is high, and preferably, it is high that sequencing result is proved to be successful rate to data redundancy, is particularly suited for FFPE groups
The ropy samples of RNA such as knit；
(2) circular rna high-throughput sequencing library structure kit of the present invention is simple to operate, condition optimizing, and low cost is ring
One powerful of shape RNA researchs.
Brief description of the drawings
Fig. 1 is a kind of flow chart of implementation method of the construction method of circular rna high-throughput sequencing library of the present invention；
Fig. 2-1 is utilized for building after the completion of storehouse for the circular rna high-throughput sequencing library in the cell sample of the embodiment of the present invention 1
Agilent 2100 carries out the result of quality inspection to library；
Fig. 2-2 be the circular rna high-throughput sequencing library in the fresh HCC tissue sample of the embodiment of the present invention 2 to build storehouse complete
Into the rear result for carrying out quality inspection to library using Agilent 2100；
Fig. 2-3 is utilized for building after the completion of storehouse for the circular rna high-throughput sequencing library in embodiment of the present invention 3FFPE samples
Agilent 2100 carries out the result of quality inspection to library；
Fig. 3 is to select 20 circular rnas after the completion of the cell sample of embodiment 1 is sequenced to carry out the result of QPCR checkings.
Specific embodiment
In order that the object, technical solutions and advantages of the present invention are clearer, the present invention is done into one below in conjunction with the accompanying drawings
Step ground is described in detail.
Specifically, as shown in figure 1, the embodiment of the present invention provides a kind of construction method of circular rna high-throughput sequencing library,
It is comprised the following steps successively：
S1, extracts the total serum IgE in sample；
The total serum IgE extracted in sample is carried out using Trizol methods, and the method is particularly well-suited to tissue and cell sample.
S2, the DNA in removal sample.
Preferably, the DNA in removal sample is carried out using DNase I digestion methods.
S3, detects and evaluates the total serum IgE quality in sample, determines RNA satisfactory qualities.
Preferably, detect and evaluate the total serum IgE quality in sample and comprise the following steps successively：
(S31) RNA palliating degradation degrees are detected using 1% agarose gel electrophoresis and whether has pollution；
(S32) using the purity of UV spectrophotometer measuring RNA, i.e. OD260/280 ratios；
(S33) integrality of RNA is detected.
The integrality of detection RNA is carried out using the biological analysers of Agilent 2100 in the step (S33).
S4, removes rRNA.
Removal rRNA is carried out using RNase H digestion methods.
RNase H digestion methods removal rRNA is comprised the following steps successively：
(S41) by mass mixings such as the DNA probes complementary with rRNA sequences, DNA probe storehouse is formed；
(S42) by obtained DNA probe storehouse with by detecting and evaluate total RNA mass in sample and determine that RNA mass is accorded with
Desired RNA bulk crossings are closed, DNA-RNA hybrids are formed；
(S43) obtained DNA-RNA hybrids are digested with RNase H；
(S44) DNA probe is digested with DNase I, obtains the RNA that rRNA exhausts；
(S45) concentration of the RNA that rRNA exhausts is determined by fluorescence photometer.
DNA probe storehouse used and the mass ratio of RNA are (1-2) in the step (S42)：1, preferably 1:1.
The condition that bulk crossing is used in the step (S42) be at a temperature of 95 DEG C hybridize 2 minutes, then with
Be slowly decreased temperature to 45 DEG C by the speed of 0.1 DEG C/s, and 45 DEG C are incubated 5 minutes.
Condition that obtained DNA-RNA hybrids are used is digested at 37 DEG C with RNase H in the step (S43)
At a temperature of digest 30 minutes.
The fluorescence photometer that the concentration of the RNA that measure rRNA exhausts is used in the step (S45) is by Qubit fluorescence photometers
Carry out.
The product obtained in the step (S45) further detects that 18S has been can't see in confirmation with the instrument of Agilent 2100,
The typical peak figure that 28S rRNA are present.
S5, removes linear rna.
Removal linear rna is carried out using RNase R digestion methods, and the amount ratio of wherein RNase R and RNA is (2.5-
3.5)U:1ug, preferably 3:1, i.e., 3 RNase R of active unit (U) are added in every 1 microgram RNA.
The digestion condition of RNase R digestion methods that removal linear rna is used at 35-45 DEG C, preferably 37 DEG C of temperature
Lower digestion 20-40 minutes, preferably 30 minutes.
S6, builds the circular rna library for high-flux sequence.
The circular rna library built for high-flux sequence is carried out using dUTP methods.
The circular rna library that dUTP methods build for high-flux sequence comprises the following steps successively：
(S61) RNA fragmentations；
(S62) the first chain synthesis：Carried out using the method for reverse transcriptase plus random primer, raw material is dNTPs (dA, dC, dG
25mM each with dT)；
(S63) the second chain synthesis：It is then poly- by DNA using the RNA chains in RNase H digestion RNA/cDNA hybrids
Synthase I synthesizes Article 2 chain, and raw material is dUTP mix (10mM dA, dC, dG and 20mM dU)；
(S64) end is repaired：The dsDNA of flat end is formed using End Repair Mix and at 5' ends plus phosphate
Group；
(S65) A tails are added：Add A tails in dsDNA ends using Klenow 3'-5'exo-；
(S66) Y types Adapter connections：Using the T4 DNA Ligase connection Adapter and dsDNA for having added A tails；
(S67) digestion of the second chain and amplified library：Using the chain of UNG enzymic digestions second；
(S68) quality inspection and preparation index library.
The condition of RNA fragmentations is at 90-96 DEG C, fragmentation 3-6 points at a temperature of preferably 94 DEG C in the step (S61)
Clock, preferably 5 minutes.
Peak value in the step (S61) after RNA fragmentations is located at 300-350bp.
The chain of step (S62) first synthesis suppresses the archaeal dna polymerase of the dependence DNA of reverse transcriptase using actinomycin D
Activity, to retain the directivity information of chain.
Adapter ends connect T using phosphorothioate bond in step (S66) the Y types Adapter connections, to prevent
Adapter dimers are formed, it is further preferred that after connection product purifying is using 1 × beads purifying once, 0.7 ×/0.2 ×
Beads carries out clip size screening, to select the fragment of suitable size.
Step (S68) quality inspection and preparation index library comprise the following steps：
(S681) Qubit fluorescence photometers detection library concentration；
(S682) Library Quality is detected using Agilent 2100；
(S683) according to library concentration, different index libraries is mixed, machine sequencing in preparation.
Compared with conventional method, the construction method of circular rna high-throughput sequencing library provided by the present invention is efficiently, surely
Fixed, rRNA residual ratios are low, and circular rna detector efficiency is high, and preferably, it is high that sequencing result is proved to be successful rate to data redundancy, especially
Suitable for ropy samples of RNA such as FFPE tissues.
In order that objects and advantages of the present invention are more concise, the present invention will be explained with specific examples below
It is bright, but the present invention is only limitted to absolutely not these embodiments.Following examples are only the present invention more preferably embodiment, and are only used for explaining
State the present invention, it is impossible to be interpreted as limiting the scope of the present invention.It should be pointed out that it is all essence of the invention and principle it
Interior done any modification, equivalent and improvement etc., should be included within the scope of the present invention.Therefore, the present invention
The protection domain of patent should be determined by the appended claims.
The structure of the circular rna high-throughput sequencing library in the cell sample of embodiment 1
(S1) extraction of the total serum IgE in cell sample
Take cell sample 1 × 107It is individual, washed once with PBS at 4 DEG C, then to adding 1mL in each hole of 6 orifice plates
Trizol, and blown and beaten repeatedly 10 times with 1mL pipette tips.By in sample collection to EP pipes, 200 μ L chloroforms, mixing of turning upside down are added
3min is stood after 30s.15min is then centrifuged at 4 DEG C of temperature and the rotating speed of 12000rpm again, solution lysate divides three layers,
It is at the middle and upper levels the RNA being soluble in the aqueous phase.Take upper solution and be added thereto to isometric isopropanol, mix and 4 DEG C of standings
10min.It is centrifuged at 4 DEG C of temperature and the rotating speed of 12000rpm again afterwards and 10min and removes supernatant.Then in precipitation
Add the ethanol of 1mL 75% and the resuspended precipitation of mixing that turns upside down.Then also at 4 DEG C of temperature and the rotating speed of 12000rpm from
Heart 10min simultaneously removes supernatant, retains precipitation.Then precipitation is dried at room temperature for 15min until tube wall no liquid.It is subsequently added
25μL DEPC H2O dissolves RNA, to obtain RNA solution.
(S2) DNA in removal cell sample
It is formulated as follows shown experimental system：
Then above-mentioned experimental system is incubated 30min at room temperature, 300 μ L Buffer RP and the mixing that is vortexed is added afterwards
15s, then stands 10min so that DNase I are inactivated.Thereafter 250 μ L absolute ethyl alcohols are added and the mixing 15s that is vortexed, is then carried out
It is of short duration to be centrifuged to remove the drop on tube wall.Then Hipure RNA pillar purifying RNAs are used, and uses 25 μ L DEPC H2O
Eluted, obtain removing the RNA sample after DNA.
(S3) detect and evaluate the total serum IgE quality in sample, determine RNA satisfactory qualities
The RNA palliating degradation degrees in the RNA sample after DNA are removed using the detection of 1% agarose gel electrophoresis and determine whether
There is pollution, as a result show that the sample is pollution-free and RNA palliating degradation degrees are small.Followed by the pure of UV spectrophotometer measuring RNA
Degree, wherein OD260/280 ratios are 1.95, and purity is higher.Analyze RNA's by the biological analysers of Agilent 2100 again afterwards
Integrality, very well, RIN values are 9.2 to display RNA integralities.
(S4) rRNA is removed
Together with the mass mixings such as 195 50bp DNA probes complementary with the rRNA sequences in cell of design, DNA is formed
Probe library, is then formulated as follows shown experimental system：
The μ g of DNA probe storehouse 5
The μ g of RNA sample 5 after removal DNA
The μ L of 5 × hybridization buffer 5 (its composition is 1M NaCl, 0.5M Tris-HCl, pH 7.4)；
Mend DEPC water to 25 μ L.
Above-mentioned experimental system is mixed two minutes at 95 DEG C, then with the speed of 0.1 DEG C/s by the temperature of experimental system
45 DEG C are reduced to, DNA-RNA hybrids are formed.Then by 10 μ L RNase H (5U/ μ L) and 5 μ L be pre-heated to 37 DEG C 10 ×
(its composition is RNase H Reaction Buffer：500mM Tris-HCl, 750mM KCL, 30mM MgCl2, 100mM
DTT, pH=8.3) and 5 μ L DEPC water add into DNA-RNA hybrids.Then digested and DNA hybridization at a temperature of 37 DEG C
RNA 30min and purified using 2.2 × RNA Clean XPbeads.Backward reaction system in add 5 μ L Turbo
DNase I (2U/ μ L) simultaneously digests DNA probe 30min under conditions of 37 DEG C.Then use 2.2 × RNA Clean XP beads
Purified, obtained the RNA of rRNA exhaustion, while determining the RNA concentration that rRNA exhausts with Qubit fluorescence photometers, as a result shown dense
It is 21.78ng/ μ L to spend.
(S5) linear rna is removed
It is formulated as follows shown experimental system：
Above-mentioned system is placed in 30min is digested in 37 DEG C of water-baths, the RNA of linear rna removal is obtained, while glimmering with Qubit
Photometry determines its concentration, and as a result display density is 4.60ng/ μ L.
(S6) the circular rna library for high-flux sequence is built
(S61) RNA fragmentations
It is formulated as follows shown experimental system：
5×First Strand Buffer 8μL
The μ L of RNA 10 of linear rna removal
Nuclease free H2O 2μL；
Above-mentioned experimental system is put into PCR instrument and is incubated, the program of the PCR instrument is：94 DEG C of 5min, are then placed at once
On ice, the RNA after fragmentation is obtained.
(S62) the first chain synthesis：
It is formulated as follows shown experimental system：
Above-mentioned experimental system is put into PCR instrument and is incubated, the program of the PCR instrument is：65 DEG C of 3min, are subsequently placed in ice
On, and add 4 μ L Nuclease free H2O、1μL 100mM DTT、0.1μL 25mM dNTPs、0.5μL SupeRase-
In, 0.5 μ L M-MulVReverse Transcriptase and 4 μ g Actinomycin D.Reaction system is then put into PCR
It is incubated in instrument, the program of the PCR instrument is：25℃10min；42℃50min；70℃15min；4℃Hold.38 μ L are added afterwards
RNA Clean XP and 19 μ L 100%ethanol are purified, and use 16 μ L Nuclease Free H2O is eluted,
RNA/cDNA hybrids are obtained, while being transferred in new pipe.
(S63) the second chain synthesis
Shown experimental system is formulated as follows at 0 DEG C：
Above-mentioned experimental system is incubated 2.5h at 16 DEG C.It is subsequently added 38 μ L RNA Clean XP and 19 μ L ethanol enters
Row purifying is simultaneously eluted with 32 μ L Qiagen EB, obtains dsDNA.
(S64) end is repaired
Shown experimental system is formulated as follows at 0 DEG C：
Above-mentioned experimental system is put into PCR instrument and at 20 DEG C 30min is kept.It is subsequently added 28 μ L RNA Clean
XP and 14 μ L ethanol are carried out and use 17 μ L Nuclease Free H2O is eluted, and obtains the dsDNA after end is repaired.
(S65) A tails are added
It is formulated as follows shown experimental system：
Then it is incubated 30min at 37 DEG C.It is subsequently added 28 μ L AMPure XP beads and 14 μ L ethanol is purified
And with 10 μ L Nuclease Free H2O is eluted, and has been added the dsDNA of A tails.
(S66) Y types Adapter connections：
Shown experimental system is formulated as follows at 0 DEG C：
Above-mentioned experimental system is put into PCR instrument and at 20 DEG C 20min is incubated, then with 24 μ L " 12P XP " beads
Mix and be incubated 6min at room temperature, afterwards reservation supernatant and by itself and 12 μ L AMPure XP beads and 5 μ L 40wt%
PEG8000 mixes, then is incubated 6min at room temperature, then with 10 μ L Nuclease Free H2O is eluted twice, is eluted
Liquid, afterwards mixes eluent with 12 μ L AMPure XP, is incubated 6min at room temperature again, then uses 30 μ L Qiagen
EB is eluted 1 time, obtains product.
(S67) digestion of the second chain and amplified library
The 15 above-mentioned products of μ L are mixed with 1 μ L Uracil DNA Glycosylase and at 37 DEG C 30min is incubated, obtained
To UNG digested DNA.Then shown experimental system is formulated as follows at 0 DEG C：
It is by the nearly PCR amplifications of above-mentioned experimental system, cycling condition：94℃30s；(98℃10s；65℃30s；72℃30s)
15 circulations of circulation；72℃5min；Then the temperature of experimental system is maintained at 4 DEG C.It is subsequently added 43 μ L AMPure XP
Beads is purified and eluted using 12 μ L Qiagen EB, obtains library.
(S68) quality inspection and preparation index library
Library concentration first is detected with Qubit fluorescence photometers, is as a result 4.06ng/ μ L.Then it is biological using Agilent 2100
Analyzer detects Library Quality, and testing result is shown in Fig. 2, as a result shows that Insert Fragment size is qualified, and peak type is single, and peak value is in 200-
500bp or so.Then according to detectable concentration, different index libraries is mixed.Opened after detecting library concentration with Qubit fluorescence photometers
Begin to continue upper machine examining order.
The structure of the circular rna high-throughput sequencing library in the fresh HCC tissue sample of embodiment 2
(S1) extraction of the total serum IgE in fresh HCC tissue sample
Fresh HCC tissue sample 50g is taken, is washed once with PBS at 4 DEG C, and shred tissue.It is subsequently added 1mL
Trizol, and be homogenized 15 times with Portable high speed disperser.Afterwards sample collection to EP is managed interior and add 200 μ L chloroforms, on
3min is stood after lower reverse mixing 30s.15min, solution cracking is then centrifuged at 4 DEG C of temperature and the rotating speed of 12000rpm again
Three layers of liquid point, it is at the middle and upper levels the RNA being soluble in the aqueous phase.Take upper solution and be added thereto to isometric isopropanol, mix simultaneously
Stand 10min.It is centrifuged at 4 DEG C of temperature and the rotating speed of 12000rpm again afterwards and 10min and removes supernatant.Then heavy
The ethanol of 1mL 75% is added in shallow lake and the resuspended precipitation of mixing that turns upside down.Then the temperature and the rotating speed of 12000rpm also at 4 DEG C
Lower centrifugation 10min simultaneously removes supernatant, retains precipitation.Then precipitation is dried at room temperature for 15min until tube wall no liquid.Then
Add 30 μ L DEPC H2O dissolves RNA, to obtain RNA solution.
(S2) DNA in removal fresh tissue sample
It is formulated as follows shown experimental system：
Then above-mentioned experimental system is incubated 30min at room temperature, 300 μ L Buffer RP and the mixing that is vortexed is added afterwards
15s, then stands 10min so that DNase I are inactivated.Thereafter 250 μ L absolute ethyl alcohols are added and the mixing 15s that is vortexed, is then carried out
It is centrifuged to remove the drop on tube wall.Then Hipure RNA pillar purifying RNAs are used, and uses 25 μ L DEPC H2O is carried out
Wash-out, obtains removing the RNA sample after DNA.
(S3) detect and evaluate the total serum IgE quality in sample, determine RNA satisfactory qualities
The RNA palliating degradation degrees in the RNA sample after DNA are removed using the detection of 1% agarose gel electrophoresis and determine whether
There is pollution, as a result show that the sample is pollution-free and RNA palliating degradation degrees are smaller.Followed by UV spectrophotometer measuring RNA's
Purity, wherein OD260/280 ratios are 2.05, and purity is higher.RNA is analyzed by the biological analysers of Agilent 2100 again afterwards
Integrality, preferably, RIN is 7.1 to display RNA integralities.
(S4) rRNA is removed
Together with the mass mixings such as 195 50bp DNA probes complementary with the rRNA sequences in flesh tissue of design, shape
Into DNA probe storehouse, shown experimental system is then formulated as follows：
The μ g of DNA probe storehouse 5
The μ g of RNA sample 5 after removal DNA
The μ L of 5 × hybridization buffer 5 (its composition is 1M NaCl, 0.5M Tris-HCl, pH7.4)
Mend DEPC water to 25 μ L.
Above-mentioned experimental system is mixed two minutes at 95 DEG C, then with the speed of 0.1 DEG C/s by the temperature of experimental system
45 DEG C are reduced to, DNA-RNA hybrids are formed.Then by 10 μ L RNase H (5U/ μ L) and 5 μ L be pre-heated to 37 DEG C 10 ×
(its composition is RNase H Reaction Buffer：500mM Tris-HCl, 750mM KCL, 30mM MgCl2, 100mM
DTT, pH=8.3) and 5 μ L DEPC water add into DNA-RNA hybrids.Then digested and DNA hybridization at a temperature of 37 DEG C
RNA 30min and purified using 2.2 × RNA Clean XP beads.Backward reaction system in add 5 μ L
Turbo DNase I (2U/ μ L) simultaneously digest DNA probe 30min under conditions of 37 DEG C.Then use 2.2 × RNA Clean XP
Beads is purified, and obtains the RNA of rRNA exhaustion, while determining the RNA concentration that rRNA exhausts with Qubit fluorescence photometers, is as a result shown
Show that concentration is 24.8ng/ μ L.
(S5) linear rna is removed
It is formulated as follows shown experimental system：
Above-mentioned system is placed in 30min is digested in 37 DEG C of water-baths, the RNA of linear rna removal is obtained, while glimmering with Qubit
Photometry determines its concentration, and as a result display density is 8.2ng/ μ L.
(S6) the circular rna library for high-flux sequence is built
(S61) RNA fragmentations
It is formulated as follows shown experimental system：
5×First Strand Buffer 8μL
The μ L of RNA 10 of linear rna removal
Nuclease free H2O 2μL；
Above-mentioned experimental system is put into PCR instrument and is incubated, the program of the PCR instrument is：94 DEG C of 5min, are then placed at once
On ice, the RNA after fragmentation is obtained.
(S62) the first chain synthesis：
It is formulated as follows shown experimental system：
Above-mentioned experimental system is put into PCR instrument and is incubated, the program of the PCR instrument is：65 DEG C of 3min, are subsequently placed in ice
On, and add 4 μ L Nuclease free H2O、1μL 100mM DTT、0.1μL 25mM dNTPs、0.5μL SupeRase-
In, 0.5 μ L M-MulVReverse Transcriptase and 4 μ g Actinomycin D.Reaction system is then put into PCR
It is incubated in instrument, the program of the PCR instrument is：25℃10min；-42℃50min；-70℃15min；-4℃Hold.Add afterwards
38 μ L RNA Clean XP and 19 μ L 100%ethanol are purified, and use 16 μ L Nuclease Free H2O is carried out
Wash-out, obtains RNA/cDNA hybrids, while being transferred in new pipe.
(S63) the second chain synthesis
Shown experimental system is formulated as follows at 0 DEG C：
Above-mentioned experimental system is incubated 2.5h at 16 DEG C.It is subsequently added 38 μ L RNA Clean XP and 19 μ L ethanol enters
Row purifying is simultaneously eluted with 32 μ L Qiagen EB, obtains dsDNA.
(S64) end is repaired
Shown experimental system is formulated as follows at 0 DEG C：
Above-mentioned experimental system is put into PCR instrument and at 20 DEG C 30min is kept.It is subsequently added 28 μ L RNA Clean
XP and 14 μ L ethanol are carried out and use 17 μ L Nuclease Free H2O is eluted, and obtains the dsDNA after end is repaired.
(S65) A tails are added
It is formulated as follows shown experimental system：
Then it is incubated 30min at 37 DEG C.It is subsequently added 28 μ L AMPure XP beads and 14 μ L ethanol is purified
And with 10 μ L Nuclease Free H2O is eluted, and has been added the dsDNA of A tails.
(S66) Y types Adapter connections：
Shown experimental system is formulated as follows at 0 DEG C：
Above-mentioned experimental system is put into PCR instrument and at 20 DEG C 20min is incubated, then with 24 μ L " 12P XP " beads
Mix and be incubated 6min at room temperature, afterwards reservation supernatant and by itself and 12 μ L AMPure XP beads and 5 μ L 40wt%
PEG8000 mixes, then is incubated 6min at room temperature, then with 10 μ L Nuclease Free H2O is eluted twice, is eluted
Liquid, afterwards mixes eluent with 12 μ L AMPure XP, is incubated 6min at room temperature again, then uses 30 μ L Qiagen
EB is eluted 1 time, obtains product.
(S67) digestion of the second chain and amplified library
The 15 above-mentioned products of μ L are mixed with 1 μ L Uracil DNA Glycosylase and at 37 DEG C 30min is incubated, obtained
To UNG digested DNA.Then shown experimental system is formulated as follows at 0 DEG C：
It is by the nearly PCR amplifications of above-mentioned experimental system, cycling condition：94℃30s；(-98℃10s；-65℃30s；-72℃
30s) circulate 15 circulations；-72℃5min；Then the temperature of experimental system is maintained at -4 DEG C.It is subsequently added 43 μ L AMPure
XP beads are purified and eluted using 12 μ L Qiagen EB, obtain library.
(S68) quality inspection and preparation index library
Library concentration first is detected with Qubit fluorescence photometers, is as a result 5.16.Then the biological analysers of Agilent 2100 are used
Detection Library Quality, testing result is shown in Fig. 2, and Insert Fragment size is qualified, and peak type is single, and peak value is in 200-500bp or so.Then
According to detectable concentration, different index libraries is mixed.Start upper machine sequencing work after detecting library concentration with Qubit fluorescence photometers
Make.
The structure of the circular rna high-throughput sequencing library in the FFPE samples of embodiment 3
(S1) extraction of the total serum IgE in FFPE samples
FFPE sample sections into the sheet of 8 μ m-thicks are taken, 7 sections to 1.5mL centrifuge tubes are shifted immediately.It is subsequently adding 1mL
Diformazan benzo is acutely vortexed 30s, and 2min is centrifuged under the rotating speed of 14000rpm.Afterwards add 1mL ethanol to sample in and whirlpool
Rotation mixes 30s, then 2min is centrifuged under the rotating speed of 14000rpm, removes supernatant, retains precipitation.Dried at a temperature of 37 DEG C
15min is removing ethanol.Then to adding 200 μ L lysates and 20 μ l Proteinase K and the mixing that is vortexed in precipitation.Thereafter
The water-bath 15min at a temperature of 55 DEG C, afterwards the water-bath 15min at a temperature of 80 DEG C.200 μ L are added to delay after the of short duration centrifugation of low speed
In fliud flushing to sample and be vortexed mixing 20s.Be subsequently added in 600 μ L absolute ethyl alcohols to samples and be vortexed mixing 20s.Shift afterwards
In mixed liquor to adsorption column, and 50s is centrifuged under the rotating speed of 8000rpm, abandons efflux, pillar in collecting pipe.With
After add 500 μ L rinsing liquids to pillars, then 50s is centrifuged under the rotating speed of 8000rpm, efflux is abandoned, pillar mounted in receiving
In collector.Thereafter in adding 500 μ L rinsing liquids to pillars, then 50s is centrifuged under the rotating speed of 8000rpm.Efflux is abandoned,
Pillar is in collecting pipe.3min then is centrifuged under the rotating speed of 13000rpm, to dry the matrix of pillar, and by posts transfer
Into new 1.5mL centrifuge tubes.30 μ L DEPC water are subsequently added to the film center of pillar and 2min, Ran Hou is stood
1min is centrifuged under the rotating speed of 13000rpm to obtain RNA solution.
(S2) DNA in removal FFPE samples
It is formulated as follows shown experimental system：
Then above-mentioned experimental system is incubated 30min at room temperature, 300 μ L Buffer RP and the mixing that is vortexed is added afterwards
15s, then stands 10min so that DNase I are inactivated.Thereafter 250 μ L absolute ethyl alcohols are added and the mixing 15s that is vortexed, is then carried out
It is centrifuged to remove the drop on tube wall.Then Hipure RNA pillar purifying RNAs are used, and uses 25 μ L DEPC H2O is carried out
Wash-out, obtains removing the RNA sample after DNA.
(S3) detect and evaluate the total serum IgE quality in sample, determine RNA satisfactory qualities
The RNA palliating degradation degrees in the RNA sample after DNA are removed using the detection of 1% agarose gel electrophoresis and determine whether
There is pollution, as a result show sample RNA obvious degradations.Followed by the purity of UV spectrophotometer measuring RNA, wherein
OD260/280 ratios are 2.37.Analyze the integrality of RNA by the biological analysers of Agilent 2100 again afterwards, RNA is complete for display
Whole property is very poor, and RIN values are 4.5.
(S4) rRNA is removed
Together with the mass mixings such as 195 50bp DNA probes complementary with the rRNA sequences in FFPE samples of design, shape
Into DNA probe storehouse, shown experimental system is then formulated as follows：
The μ g of DNA probe storehouse 5
The μ g of RNA sample 5 after removal DNA
The μ L of 5 × hybridization buffer 5 (its composition is 1M NaCl, 0.5M Tris-HCl, pH 7.4)
Mend DEPC water to 25 μ L.
Above-mentioned experimental system is mixed two minutes at 95 DEG C, then with the speed of 0.1 DEG C/s by the temperature of experimental system
45 DEG C are reduced to, DNA-RNA hybrids are formed.Then by 10 μ L RNase H (5U/ μ L) and 5 μ L be pre-heated to 37 DEG C 10 ×
(its composition is RNase H Reaction Buffer：500mM Tris-HCl, 750mM KCL, 30mM MgCl2, 100mM
DTT, pH=8.3) and 5 μ L DEPC water add into DNA-RNA hybrids.Then digested and DNA hybridization at a temperature of 37 DEG C
RNA 30min and purified using 2.2 × RNA Clean XP beads.Backward reaction system in add 5 μ L
Turbo DNase I (2U/ μ L) simultaneously digest DNA probe 30min under conditions of 37 DEG C.Then use 2.2 × RNA Clean XP
Beads is purified, and obtains the RNA of rRNA exhaustion, while determining the RNA concentration that rRNA exhausts with Qubit fluorescence photometers, is as a result shown
Show that concentration is 19.9ng/ μ L.
(S5) linear rna is removed
It is formulated as follows shown experimental system：
Above-mentioned system is placed in 30min is digested in 37 DEG C of water-baths, the RNA of linear rna removal is obtained, while glimmering with Qubit
Photometry determines its concentration, and as a result display density is 3.25ng/ μ L.
(S6) the circular rna library for high-flux sequence is built
(S61) RNA fragmentations
It is formulated as follows shown experimental system：
5×First Strand Buffer 8μL
The μ L of RNA 10 of linear rna removal
Nuclease free H2O 2μL；
Above-mentioned experimental system is put into PCR instrument and is incubated, the program of the PCR instrument is：94 DEG C of 5min, are then placed at once
On ice, the RNA after fragmentation is obtained.
(S62) the first chain synthesis：
It is formulated as follows shown experimental system：
Above-mentioned experimental system is put into PCR instrument and is incubated, the program of the PCR instrument is：65 DEG C of 3min, are subsequently placed in ice
On, and add 4 μ L Nuclease free H2O、1μL 100mM DTT、0.1μL 25mM dNTPs、0.5μL SupeRase-
In, 0.5 μ L M-MulVReverse Transcriptase and 4 μ g Actinomycin D.Reaction system is then put into PCR
It is incubated in instrument, the program of the PCR instrument is：25℃10min；-42℃50min；-70℃15min；-4℃Hold.Add afterwards
38 μ L RNA Clean XP and 19 μ L 100%ethanol are purified, and use 16 μ L Nuclease Free H2O is carried out
Wash-out, obtains RNA/cDNA hybrids, while be transferred in new pipe,.
(S63) the second chain synthesis
Shown experimental system is formulated as follows at 0 DEG C：
Above-mentioned experimental system is incubated 2.5h at 16 DEG C.It is subsequently added 38 μ L RNA Clean XP and 19 μ L ethanol enters
Row purifying is simultaneously eluted with 32 μ L Qiagen EB, obtains dsDNA.
(S64) end is repaired
Shown experimental system is formulated as follows at 0 DEG C：
Above-mentioned experimental system is put into PCR instrument and at 20 DEG C 30min is kept.It is subsequently added 28 μ L RNA Clean
XP and 14 μ L ethanol are carried out and use 17 μ L Nuclease Free H2O is eluted, and obtains the dsDNA after end is repaired.
(S65) A tails are added
It is formulated as follows shown experimental system：
Then it is incubated 30min at 37 DEG C.It is subsequently added 28 μ L AMPure XP beads and 14 μ L ethanol is purified
And with 10 μ L Nuclease Free H2O is eluted, and has been added the dsDNA of A tails.
(S66) Y types Adapter connections：
Shown experimental system is formulated as follows at 0 DEG C：
Above-mentioned experimental system is put into PCR instrument and at 20 DEG C 20min is incubated, then with 24 μ L " 12P XP " beads
Mix and be incubated 6min at room temperature, afterwards reservation supernatant and by itself and 12 μ L AMPure XP beads and 5 μ L 40wt%
PEG8000 mixes, then is incubated 6min at room temperature, then with 10 μ L Nuclease Free H2O is eluted twice, is eluted
Liquid, afterwards mixes eluent with 12 μ L AMPure XP, is incubated 6min at room temperature again, then uses 30 μ L Qiagen
EB is eluted 1 time, obtains product.
(S67) digestion of the second chain and amplified library
The 15 above-mentioned products of μ L are mixed with 1 μ L Uracil DNA Glycosylase and at 37 DEG C 30min is incubated, obtained
To UNG digested DNA.Then shown experimental system is formulated as follows at 0 DEG C：
It is by the nearly PCR amplifications of above-mentioned experimental system, cycling condition：94℃30s；(98℃10s；65℃30s；72℃30s)
15 circulations of circulation；72℃5min；Then the temperature of experimental system is maintained at 4 DEG C.It is subsequently added 43 μ L AMPure XP
Beads is purified and eluted using 12 μ L Qiagen EB, obtains library.
(S68) quality inspection and preparation index library
Library concentration first is detected with Qubit fluorescence photometers, is as a result 5.24ng/ μ L.Then it is biological using Agilent 2100
Analyzer detects Library Quality, and testing result is shown in Fig. 2, and Insert Fragment size is qualified, and peak type is single, and peak value is left in 200-500bp
It is right.Then according to detectable concentration, different index libraries is mixed.Start on continuous after detecting library concentration with Qubit fluorescence photometers
Machine examining order.
Sequencing embodiment bioinformatic analysis sequencing data
The circular rna high-throughput sequencing library and literature procedure that embodiment 1-3 is built are (referring to Molecular
Cell 56,55-66, October 2,2014) build circular rna high-throughput sequencing library enter in Illumina microarray datasets
Row sequencing.Result is shown in Table 1.
The inventive method of table 1 and literature procedure sequencing result bioinformatic analysis
Sequencing data the results are shown in Table 1, under contrast, side provided in an embodiment of the present invention through after bioinformatic analysis
Method can further remove the rRNA in sample, rRNA residuals ratio in data<0.1%, and rRNA in literature procedure
It is 5% to remain, and the RNA sample serious for the degraded such as FFPE is more up to 37%, and serious waste data volume, the inventive method makes
The effective sequencing data amount for obtaining circular rna is significantly improved.In terms of circular rna amount detection, the good sample of RNA integralities is (thin
Born of the same parents' sample) it is more with the circular rna of literature procedure detection, but the ropy samples of RNA, especially FFPE sample copies hair
Bright method is significantly better than literature procedure.Additionally, circular rna high-throughput sequencing library structure is carried out using the inventive method,
There is the operating process of standard, as a result condition optimizing is stablized, cost is only the 1/3 of literature procedure.It is random from sequencing result
Selecting 20 circular rnas carries out QPCR checkings, the result positive rate 100% (Fig. 3), and effectively confirm the inventive method can
By property.
Thus significantly further can be enriched with the circular rna in sample by the method used by the provable embodiment of the present invention,
Circular rna sequencing data amount is greatly lowered, reduces cost reduces the waste of manpower and materials.
The announcement and teaching of book according to the above description, those skilled in the art in the invention can also be to above-mentioned embodiment party
Formula is changed and changed.Therefore, the invention is not limited in specific embodiment disclosed and described above, to the one of invention
A little modifications and changes should also be as falling into scope of the claims of the invention.Although additionally, being used in this specification
Some specific terms, but these terms are merely for convenience of description, do not constitute any limitation to the present invention.
Claims (9)
1. a kind of construction method of circular rna high-throughput sequencing library, it is comprised the following steps successively：
(S1) total serum IgE in sample is extracted；
(S2) DNA in removal sample；
(S3) detect and evaluate the total serum IgE quality in sample, determine RNA satisfactory qualities；
(S4) rRNA is removed；
(S5) linear rna is removed；
(S6) the circular rna library for high-flux sequence is built.
2. construction method according to claim 1, it is characterised in that RNA total in detection sample in the step (S3)
Quality is comprised the following steps successively：
(S31) RNA palliating degradation degrees are detected using 1% agarose gel electrophoresis and whether has pollution；
(S32) using the purity of UV spectrophotometer measuring RNA, i.e. OD260/280 ratios；
(S33) integrality of RNA is detected.
3. construction method according to claim 1, it is characterised in that step (S4) the removal rRNA includes following successively
Step：
(S41) by mass mixings such as the DNA probes complementary with rRNA sequences, DNA probe storehouse is formed；
(S42) obtained DNA probe storehouse is detected and evaluates the total serum IgE quality in sample with process and determine that RNA mass is conformed to
The RNA bulk crossings asked, form DNA-RNA hybrids；
(S43) obtained DNA-RNA hybrids are digested with RNase H；
(S44) DNA probe is digested with DNase I, obtains the RNA that rRNA exhausts；
(S45) concentration of the RNA that rRNA exhausts is determined by fluorescence photometer.
4. construction method according to claim 3, it is characterised in that in the step (S42) DNA probe storehouse used with
The weight ratio of RNA is (1-2)：1, preferably 1:1.
5. construction method according to claim 3, it is characterised in that bulk crossing is used in the step (S42)
Condition is to hybridize 2 minutes at a temperature of 95 DEG C, and temperature is slowly decreased to 45 DEG C with the speed of 0.1 DEG C/s then.
6. construction method according to claim 3, it is characterised in that digested with RNase H in the step (S43) and obtained
The condition that the DNA-RNA hybrids for obtaining are used is to be digested 30 minutes at a temperature of 37 DEG C.
7. construction method according to claim 1, it is characterised in that built for high-flux sequence in the step (S6)
Circular rna library comprise the following steps successively：
(S61) RNA fragmentations；
(S62) the first chain synthesis：Carried out using the method for reverse transcriptase plus random primer；
(S63) the second chain synthesis：Using the RNA chains in RNase H digestion RNA/cDNA hybrids, then by DNA polymerase i
Synthesis Article 2 chain；
(S64) end is repaired：The dsDNA of flat end is formed using End Repair Mix and at 5' ends plus phosphate group；
(S65) A tails are added：Add A tails in dsDNA ends using Klenow 3'-5'exo-；
(S66) Y types Adapter connections：Using the T4DNA Ligase connection Adapter and dsDNA for having added A tails；
(S67) digestion of the second chain and amplified library：Using the chain of UNG enzymic digestions second；
(S68) quality inspection and preparation index library.
8. construction method according to claim 7, it is characterised in that the condition of RNA fragmentations is in the step (S61)
At 90-96 DEG C, fragmentation 3-6 minutes at a temperature of preferably 94 DEG C, preferably 5 minutes.
9. the kit that a kind of circular rna high-throughput sequencing library builds, it is characterised in that it includes：
(1) circular rna enrichment reagents, DNase I, RNase H, RNaseR；
(2) the special DNA probe of kind；
(3) the various enzymes needed for library construction：DNA polymerase i, Large Fragment, T4Polynucleotide
Kinase、Klenow Fragment、Escherichia coli UDG、T4DNA Ligase、Phusion High-
Fidelity DNA Polymerase；
(4) magnetic bead, dNTP, Nuclease Free Water, dUTP, joint, primer,
Wherein, the special DNA probe of the kind can be complementary with the rRNA sequences of corresponding species.
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| CN106801050B (en) | 2020-01-14 |
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Effective date of registration: 20201130 Address after: 510300 Guangzhou Development Zone, Guangdong Province, No. 6, Spiral Third Road, Guangzhou International Biological Island, 4th Floor Patentee after: GUANGZHOU FOREVERGEN BIOTECHNOLOGY Co.,Ltd. Patentee after: GUANGZHOU FOREVERGEN MEDICAL LABORATORY Co.,Ltd. Address before: 2, building 602, building 8, No. three, No. 510300, Guangzhou International Biological Island, Guangzhou Development Zone, Guangdong, China Patentee before: GUANGZHOU FOREVERGEN BIOTECHNOLOGY Co.,Ltd. Patentee before: GUANGZHOU FOREVERGENDX TECHNOLOGY Co.,Ltd. |
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