Method for constructing strand-specific transcriptome library

A transcriptome-specific technology, applied in the field of constructing strand-specific transcriptome libraries, can solve the problems of low labeling efficiency at the 3' end of cDNA, facilitate data analysis, improve cDNA synthesis efficiency, and reduce the production of linker dimers. Effect

Inactive Publication Date: 2016-02-24
生工生物工程(上海)股份有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The present invention aims at the technical problem of low cDNA 3' end labeling efficiency in the prior

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  • Method for constructing strand-specific transcriptome library
  • Method for constructing strand-specific transcriptome library
  • Method for constructing strand-specific transcriptome library

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] mRNA fragmentation treatment: Qualified RNA was extracted, and mRNA was purified by mixing 2550 μL of Dynabeadsoligo (dT) from life-tech company with 1 μg of total RNA. Take 7 μL of 5× (that is, 5 times) fragmentbuffer and add it to 28 μL of mRNA, 94°C for 5 minutes, and then put it on ice immediately. Add 65 μLddH 2 O (i.e. double distilled water), 20 μg glycogen, 1 / 10 volume of 3M pH5.2 NaAc, 2.5 times volume of absolute ethanol, mix well, store at -80°C for 20 min; centrifuge at high speed, and dissolve the precipitate in 13 μL deionized water.

[0028] Ligation of R3 linker: mRNA dissolved in 5 μL ddH2 In O, add 1 μL of 10 μM R3 linker, 70 ° C for 2 min, and place on ice for 1 min. Add 1.5μL ligationMix, 200UT4 truncated RNA ligase 2K227Q (T4RNAligase2TruncatedK227Q), ddH 2 Make up to 15 μL with O, mix well, and keep at 22°C for 1h.

[0029] cDNA synthesis: Add 1 μL of 50 μM RTprimer, 65°C for 5 min, and place on ice for 1 min; add 4 μL of RTMix, 20 UMMLV reverse...

Embodiment 2

[0034] mRNA fragmentation treatment: Qualified RNA was extracted, and mRNA was purified by mixing 2530 μL of Dynabeadsoligo (dT) from life-tech company with 1 μg of total RNA. Take 7 μL of 5×fragmentbuffer and add it to 28 μL of mRNA, 94°C for 5 minutes, and then put it on ice immediately. Add 65 μLddH 2 O, 20 μg glycogen, 1 / 10 volume of 3M NaAc at pH 5.2, 2.5 volumes of absolute ethanol, mix well, store at -80°C for 20 min; centrifuge at high speed, and dissolve the precipitate in 13 μL deionized water.

[0035] Ligation of R3 linker: mRNA dissolved in 5 μL ddH 2 In O, add 0.5 μL of 10 μM R3 adapter, 70 ° C for 2 min, and place on ice for 1 min. Add 1.5 μL of ligationMix, 200UT4RNAligase2TruncatedK227Q, ddH 2 Make up to 15 μL with O, mix well, and keep at 22°C for 1h.

[0036] cDNA synthesis: Add 0.5 μL of 50 μM RTprimer, 65° C. for 5 minutes, and place on ice for 1 minute. Add 4 μL RTMix, 20 UMMLV reverse transcriptase, ddH 2 Make up to 20ul with O, mix well, then stor...

Embodiment 3

[0046] mRNA fragmentation treatment: Qualified RNA was extracted, and mRNA was purified by mixing 2550 μL of Dynabeadsoligo (dT) from life-tech company with 1 μg of total RNA. Take 7 μL of 5Xfragmentbuffer and add to 28 μL of mRNA, 94°C for 5 minutes, and then put it on ice immediately. Add 65 μLddH 2 O, 20 μg glycogen, 1 / 10 volume of 3M pH5.2NaAc, 2.5 times the volume of absolute ethanol, mix well, store at -80°C for 20min; centrifuge at high speed, and dissolve the precipitate in 13 μL deionized water.

[0047] Ligation of R3 linker: mRNA dissolved in 5 μL ddH 2 In O, add 1 μL of 10 μM R3 linker, 70 ° C for 2 min, and place on ice for 1 min. Add 1.5 μL of ligationMix, 200UT4RNAligase2TruncatedK227Q, ddH 2 Make up to 15 μL with O, mix well, and keep at 22°C for 1h.

[0048] cDNA synthesis: Add 1 μL of 50 μM RTprimer, 65°C for 5 min, and place on ice for 1 min; add 4 μL of RTMix, 20 UMMLV reverse transcriptase, ddH 2 Make up to 20 μL with O, mix well, then store at 25°C f...

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Abstract

The invention discloses a method for constructing a strand-specific transcriptome library. The method comprises steps as follows: step 1), mRNA (massager ribonucleic acid) fragmentation processing is performed; step 2), inverse transcription is performed: the fragmented mRNA 3' terminal is connected with an R3 joint with the known sequence; an RT primer and the R3 joint are subjected to annealing pairing for inverse transcription, and a first strand of cDNA (complementary desoxyribonucleic acid) is synthesized; step 3), cDNA 3' terminal marking is performed: after RNA is removed, TdT (terminal deoxynucleotidyl transferase) is adopted to add multiple dC basic groups to the cDNA 3' terminal, ddCTP (2',3'-dideoxycytidine-5'-triphosphate) is adopted for end closing, pairing is performed with an F5G primer, and the first strand of cDNA is duplicated under the action of polymerase; step 4), PCR (polymerase chain reaction) amplification is performed; step 5), quality testing is performed. The fragmented mRNA is connected with the joint and then is synthesized, so that the synthetic efficiency and the homogeneity of cDNA are improved; after the first strand of cDNA is synthesized, multiple dC basic groups are added to the cDNA 3' terminal by adopting TdT and ddCTP is adopted for closing, pairing is performed with the F5G primer containing multiple dG basic groups, the first strand of cDNA is duplicated under the action of polymerase, therefore, production of self-linked dimers can be effectively reduced, and the homogeneity of the library is improved.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for constructing a strand-specific transcriptome library. Background technique [0002] As a newly developed high-throughput sequencing technology in recent years, next-generation sequencing of transcriptome provides a new and more effective method for large-scale transcriptomics research. This technology can comprehensively and quickly obtain almost all transcripts of a specific organ or tissue of a species in a certain state at the single nucleotide level. Compared with other transcriptomic technologies, it has obvious advantages such as high throughput, low cost, and high sensitivity, and can obtain low-abundance expressed genes. [0003] At present, major biological companies and literatures have reported a variety of library construction methods: from the initial need for full-length mRNA for library construction to the current use of fragme...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68C40B50/06
CPCC12N15/1096C12N2330/31C12Q1/68C40B50/06
Inventor 杨吉元
Owner 生工生物工程(上海)股份有限公司
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