1 After miRNA cleavage, the mRNA produces two fragments at the tenth or eleventh base, the first fragment is the 5' cap structure and the 3' carboxyl group. The 3' cut fragment contains a free 5' monophosphate and 3' poly(A) tail.
2 The 5' adapter can be used to connect to the RNA of the 5' monophosphate group, but the group lacking 5' monophosphate cannot be connected to the cleavage product of the captured miRNA, and then the 5'- The 3' cleavage product of the adaptor was reverse transcribed into cDNA. After the cDNA was amplified, the PCR product was digested with the restriction endonuclease Mme I with a length of 20-21 bp. The digested 3' double-stranded DNA adaptor was ligated and amplified by PCR. After the product was gel purified, a library containing the 3' end cut fragments cut by miRNA was established for sequencing.