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Method for capturing RNA in-situ advanced structure and interaction
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A high-level structure and in-situ technology, applied in the biological field, can solve the problems of excessive useless data, inability to capture, and low ratio
Active Publication Date: 2020-11-10
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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Problems solved by technology
Although the above methods can study the single-stranded and double-stranded regions of RNA with high throughput, they also have some defects: First, they cannot capture non-Watson-Crick pairings and long-distance RNAloop-loop interactions
Second, these ligation reactions are all carried out in solution, and there are non-specific connections, which cannot reflect the real structure of RNA in the cell, resulting in a large number of false positive intermolecular connections
Third, in the data obtained by sequencing, the ratio of chimeric reads (chimeric reads, that is, the products connected between different RNA fragments) is low, and there are too many useless data
CHIRP, CHART and RAP methods only focus on DNA targets, ignoring RNA target sites with important functions, and can only identify all potential DNA targets (one to all) of one lncRNA in cells at a time, and the throughput is too low
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Embodiment 1
[0117] Embodiment 1, the preparation method of RIC-seq library
[0118] The RIC-seq library construction process of the present invention is as follows figure 1 Shown in A. These include cultured cells, formaldehyde crosslinking, cell membrane and nuclear membrane perforation, MNase enzyme treatment, RNA 3' end hydroxylation, pCp-biotin ligation, RNA 3' end hydroxylation, 5' end phosphorylation, proximal ligation , total RNA extraction, DNase I removal of genomic DNA, ribosomal RNA removal, RNA fragmentation, C1 magnetic bead enrichment and elution of enriched RNA, cDNA first-strand synthesis, DNA second-strand synthesis, end repair, adding "A" , link adapter, PCR amplification and other steps. Specific steps are as follows:
[0119] 1. Take cells in a 15 cm plate with a density of about 80-90%, discard the medium, add 10 ml of pre-cooled PBS (pH 7.4) to wash the cells, discard the PBS, and repeat this step 3 times to obtain washed cells.
[0120] 2. After completing step ...
Embodiment 2
[0152] Example 2, Application of the preparation method of RIC-seq library
[0153] 1. Culture HeLa cells and Drosophila S2 cell samples
[0154] HeLa cells cultured in the laboratory were used as samples, and the initial cell sample volume was 1×10 7 Individual cells, Drosophila S2 cells serving as spike-in, were assessed for proximal junction specificity.
[0155] 2. Preparation of RIC-seq library
[0156] Based on the cell sample in step 1, a RIC-seq library was constructed according to the method in Example 1. Wherein, the upstream and downstream primers in step 30 are as follows respectively (NNNNNNN is the library Index sequence)
[0157] Primer1.0
[0158] 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID No. 1);
[0159] Index primer
[0160] 5'-CAAGCAGAAGACGGCATACGAGATANNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3' (SEQ ID No. 2).
[0161] Wherein, N represents A or T or C or G.
[0162] 3. Sequencing
[0163] PE150 paired-end sequencing ...
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Abstract
The invention discloses a method for capturing an RNA in-situ advanced structure and interaction. The method comprises the following steps of immobilizing protein-mediated RNA interaction in cells ortissue; performing membrane perforation under the condition of keeping cells intact; degrading free RNA; performing pCp-biotin labeling at the tail end of RNA3' and performing in-situ near-end connection; purifying chimeric RNA containing C-biotin after the cells are digested; constructing a chain specificity library; and performing high throughput sequencing. According to the method, intracellular RNA is treated in situ under the conditions of not damaging the cell structure and keeping the cells intact, and RNA intramolecular (intermolecular) interaction in a physiological state is captured;PCp-biotin is used for labelling the tail end of RNA, in-situ connection is performed under the non-denaturation condition, the labelling efficiency is greatly improved, and besides, intermolecular non-specific connection is reduced; and the chimeric RNA of the labeled C-biotin can be efficiently enriched by using C1 magnetic beads, so that the effective data proportion of sequencing is increased, and the sequencing cost is reduced.
Description
technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for capturing RNA in-situ high-level structure and interaction. Background technique [0002] DNA, the carrier of genetic information, needs to be transcribed into RNA first, and then translated into protein to exert biological functions. As a carrier of genetic information, RNA mainly functions to encode and guide protein synthesis. This type of protein-encoding RNA is collectively called messenger RNA (messenger RNA, mRNA). In addition, the human genome also transcribes and produces a large number of RNAs that do not encode proteins, and this type of RNA is called noncoding RNA (noncoding RNA, ncRNA). The non-coding RNAs that have been discovered so far include: tRNA, rRNA, siRNA, miRNA, piRNA, snoRNA, circRNA, and lncRNA, etc. Their abnormal expression and mutation are associated with major diseases such as cancer, development, and reproductive defects. As a...
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