Strand displacement primer and cell transcriptome library construction method
A library construction and transcriptome technology, applied in biochemical equipment and methods, chemical libraries, combinatorial chemistry, etc., can solve the problems of inconvenient labeling, inability to compare RNA content, limitations, etc., and achieve the effect of sensitive amplification
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Embodiment 1
[0066] Example 1: Cell labeling and cell lysis
[0067] The method disclosed in the present invention starts with a single cell or a small number of cells. First, single cells are isolated by flow cytometry or operated under a microscope with the naked eye, and the single cells are placed in a pre-configured lysate. Due to the small amount of cells, only a small amount of lysate is needed to completely lyse the cells. At the same time, due to the small amount, it is necessary to add DNA probes, RNase inhibitors and glycogen to the lysate. As the lysate, RLT plus lysate from Qiagen was selected. The amount of glycogen is 0.5ul-1.5ul, preferably 1ul per reaction (or cell), and the amount of RNase inhibitor is 1U-3U / ul, preferably 2U / ul. An optional operation is to add 20ul of ethanol and 5ul of protease solution (Qiagen) after the cells are placed in the lysate. RNA is further released by protease digestion in lysate, followed by alcohol precipitation to concentrate the reac...
Embodiment 2
[0068] Example 2: DNA Removal and RNA Fragmentation
[0069] In order to minimize interference, the genomic DNA of cells was removed before library construction. Due to the characteristics of next-generation sequencing, for Illumina sequencers, the length of inserted RNA needs to be controlled below 300bp in order to obtain as much as possible all RNA information. Dissolve the RNA precipitate obtained in the previous step in 9ul of 1X PFE buffer (2X PFE buffer, TaKaRa), and add 0.5ul DNase I, remove the genome at 42°C, and then react at 85°C for 5-6 minutes, preferably 5 min 30 sec to allow RNA fragmentation. DNase I was then inactivated at 75°C for 10 minutes.
Embodiment 3
[0070] Example 3: RNA 3' end repair and ligation
[0071] Add T4 PNK and PNK buffer directly to the above reaction system, and react at 37°C for 30 minutes to 60 minutes, preferably 45 minutes, to remove the phosphate at the 3′ end of the RNA to obtain repaired RNA with a hydroxyl group at the 3′ end. Add pre-configured adapters to the end-repaired RNA system, the concentration of the adapters is 1uM, and add 0.5ul-1ul adapters to each reaction (cell), preferably 1ul. Add T4 RNA ligase (NEB, T4 RNA ligase truncked KQ) and incubate the reaction at 4°C with shaking for 18-24 hours, preferably 24 hours. Alternatively the ligation reaction was incubated at 20°C for 4 hrs, 4 hrs for 2 hrs.
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