In this project, the mRNA from NmrA-like gene knock out EBL cells, characterized by a polyA structure within the total RNA, was enriched using Oligo(dT) magnetic beads. Subsequently, ion interruption was employed to fragment the RNA into segments approximately 300 base pairs in length. The fragment with a length of 300bp was chosen because the length of the junction is fixed, and if the length of the interrupted fragment is short, it will lead to a high proportion of the junction sequence, which will reduce the proportion of valid data; if the length of the interrupted fragment is long, it will be unfavorable to the generation of clusters in the process of on-line sequencing. The first strand of cDNA was synthesized using RNA as template with 6-base random primer and reverse transcriptase, and the first strand cDNA was used as template for the second strand cDNA synthesis.