Method for constructing mRNA 5'-termnal information library

Abstract

The invention belongs to the technical field of genomics, and particularly relate to a method for simplifying CAGE-seq library establishing.The method comprises the steps that mRNA and lncRNA containing cap structures are enriched, a 5'-termnal connector is specifically added, after fragmentation processing, reverse transcription is carried out, and 5'-terminal information is further enriched.According to the whole method, an existing mature RNA-seq kit of the gnomegen company is mixed.The method is easier to operate and high in success rate, the 5'-terminal information can be specifically enriched, the method is more helpful for verifying the transcriptional start site of mRNA in a whole cell, and thus acquiring accurate promoter information and 5'UTR information.The method is a powerful mode for studying gene expressions and is suitable for gene regulatory network studies.

Description

technical field

[0001] The invention belongs to the field of molecular biology, in particular to a method for constructing an mRNA 5' end information library. Background technique

[0002] The most important mode of gene expression regulation is transcription regulation, that is, the regulation of interaction between promoter cis-acting elements and transcription machinery. The core promoter located upstream of the transcription start site (TSS) contains the most important elements that interact with the transcription machinery, so the accurate identification of the promoter is crucial for studying the regulation of gene expression. Different transcription start sites will cause genes to use different 5'UTRs. The 5'UTR is critical to the initiation of translation of mRNA and is one of the most important target regions for the regulation of translation efficiency. The vast majority of genes have more than two transcription initiation sites. Systematically identifying the tr...

Examples

Embodiment 1

[0039] [Example 1] Capturing samples

[0040] Take 20 μg of RNA and add DNaseI enzyme (promega) for digestion to digest the DNA in the RNA sample to prevent genome contamination. Purified RNA, use 60μl Oligo(dT)25 calls up mRNA with a polyA tail. The step of purifying mRNA by magnetic beads can follow the standard procedure provided by Ambion. The sample after two rounds of elution was redissolved in 20 μl of 10 mM Tris-HCl.

[0041] Here are the detailed steps:

[0042] 1.1. Prepare the water bath and thermomixer.

[0043] 1.2. After heating the RNA sample in a water bath at 65°C for 5 minutes (to destroy the secondary structure), put it on ice quickly.

[0044] 1.3. Take 60μl Oligo(dT)25 into a new RNase-free EP tube.

[0045] 1.4. Add 100 μl BeadBindingBuffer and gently vortex to mix and stir for 1 minute, flash shake, put it on the magnetic stand, discard the supernatant, wash the beads again with BeadBindingBuffer, and remove the supernatant.

[0046] 1.5. Take 5...

Embodiment 2

[0055] [Example 2] The 5' end cap structure is connected to the 5' connector

[0056] 2.1 Dephosphorylation of RNA not protected by cap structure

[0057] First, select fastAP (NEB) enzyme, the reaction system is as follows,

[0058]

[0059] 2.2 use Oligo(dT)25 Purified Nucleic Acid

[0060] After the reaction is complete, use Oligo(dT)25 purifies nucleic acid and removes salt ions and enzymes in the reaction system. The reaction steps are as follows:

[0061] 2.2.1. Vortex the beads to mix well, take 60μl of beads and place them in a 1.5ml centrifuge tube;

[0062] 2.2.2. Take 100 μl of BindingBuffer and vortex to wash the magnetic beads, discard the supernatant for later use, repeat once, then take 50 μl of BindingBuffer to resuspend the magnetic beads;

[0063] 2.2.3. Rehydrate the sample obtained in 2.1 above to 50 μl, add it to 50 μl beads, mix by pipetting or vortexing, and incubate on a rotary mixer for 5 minutes;

[0064] 2.2.4. After incubation for 5 minut...

Embodiment 3

[0085] [Example 3] cDNA synthesis by reverse transcription

[0086] For the library construction process, refer to the RNA-seqkit operation manual of gnomegen company.

[0087] The sample was fragmented and incubated at 95°C for 5 min, and then the fragmented sample was purified with GnomeSizeSelector. Rehydrate the purified fragmented product to 40 μl, add 24 μl GnomeSizeSelector and pipette 5 times to mix, then add 16 μl 100% Ethanol, pipette 5 times to mix. Stand at room temperature for 5 minutes. Transfer the sample to a magnetic stand and let it stand for 3 minutes. Discard the supernatant. Put the sample on the magnetic stand, add 100 μl of 70% Ethanol and pipette for 3 times to wash. Absorb the 70% Ethanol cleanly, remove the sample from the magnetic stand, and let it dry for 5 minutes. Take 10 μl of DEPC water for each sample to redissolve, and pipette 5 times to resuspend the magnetic beads. Rotate the sample at a slow speed for 2 minutes, then place it on the m...

technical field

[0001] The invention belongs to the field of molecular biology, in particular to a method for constructing an mRNA 5' end information library. Background technique

[0002] The most important mode of gene expression regulation is transcription regulation, that is, the regulation of interaction between promoter cis-acting elements and transcription machinery. The core promoter located upstream of the transcription start site (TSS) contains the most important elements that interact with the transcription machinery, so the accurate identification of the promoter is crucial for studying the regulation of gene expression. Different transcription start sites will cause genes to use different 5'UTRs. The 5'UTR is critical to the initiation of translation of mRNA and is one of the most important target regions for the regulation of translation efficiency. The vast majority of genes have more than two transcription initiation sites. Systematically identifying the tr...

Examples

Embodiment 1

[0039] [Example 1] Capturing samples

[0040] Take 20 μg of RNA and add DNaseI enzyme (promega) for digestion to digest the DNA in the RNA sample to prevent genome contamination. Purified RNA, use 60μl Oligo(dT)25 calls up mRNA with a polyA tail. The step of purifying mRNA by magnetic beads can follow the standard procedure provided by Ambion. The sample after two rounds of elution was redissolved in 20 μl of 10 mM Tris-HCl.

[0041] Here are the detailed steps:

[0042] 1.1. Prepare the water bath and thermomixer.

[0043] 1.2. After heating the RNA sample in a water bath at 65°C for 5 minutes (to destroy the secondary structure), put it on ice quickly.

[0044] 1.3. Take 60μl Oligo(dT)25 into a new RNase-free EP tube.

[0045] 1.4. Add 100 μl BeadBindingBuffer and gently vortex to mix and stir for 1 minute, flash shake, put it on the magnetic stand, discard the supernatant, wash the beads again with BeadBindingBuffer, and remove the supernatant.

[0046] 1.5. Take 5...

Embodiment 2

[0055] [Example 2] The 5' end cap structure is connected to the 5' connector

[0056] 2.1 Dephosphorylation of RNA not protected by cap structure

[0057] First, select fastAP (NEB) enzyme, the reaction system is as follows,

[0058]

[0059] 2.2 use Oligo(dT)25 Purified Nucleic Acid

[0060] After the reaction is complete, use Oligo(dT)25 purifies nucleic acid and removes salt ions and enzymes in the reaction system. The reaction steps are as follows:

[0061] 2.2.1. Vortex the beads to mix well, take 60μl of beads and place them in a 1.5ml centrifuge tube;

[0062] 2.2.2. Take 100 μl of BindingBuffer and vortex to wash the magnetic beads, discard the supernatant for later use, repeat once, then take 50 μl of BindingBuffer to resuspend the magnetic beads;

[0063] 2.2.3. Rehydrate the sample obtained in 2.1 above to 50 μl, add it to 50 μl beads, mix by pipetting or vortexing, and incubate on a rotary mixer for 5 minutes;

[0064] 2.2.4. After incubation for 5 minut...

Embodiment 3

[0085] [Example 3] cDNA synthesis by reverse transcription

[0086] For the library construction process, refer to the RNA-seqkit operation manual of gnomegen company.

[0087] The sample was fragmented and incubated at 95°C for 5 min, and then the fragmented sample was purified with GnomeSizeSelector. Rehydrate the purified fragmented product to 40 μl, add 24 μl GnomeSizeSelector and pipette 5 times to mix, then add 16 μl 100% Ethanol, pipette 5 times to mix. Stand at room temperature for 5 minutes. Transfer the sample to a magnetic stand and let it stand for 3 minutes. Discard the supernatant. Put the sample on the magnetic stand, add 100 μl of 70% Ethanol and pipette for 3 times to wash. Absorb the 70% Ethanol cleanly, remove the sample from the magnetic stand, and let it dry for 5 minutes. Take 10 μl of DEPC water for each sample to redissolve, and pipette 5 times to resuspend the magnetic beads. Rotate the sample at a slow speed for 2 minutes, then place it on the m...

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