GSM7445311: HNC_3; Homo sapiens; OTHER
Cell lysates and polyriboTRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract the mRNA from the cell lysate and the mRNA bound to the polysomes. RNA dissolved in DEPC-treated ddH2O.some bound mrnas were extracted by TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The Agilent 2200 TapeStation Nucleic Acid System was employed to detect the RNA integrity number (RIN) and concentration of RNA samples. To construct the cDNA library, RNA samples with a RIN greater than 8.5 were used. A certain amount of RNA was taken, denatured at an appropriate temperature to open its secondary structure, and the mRNA was enriched using oligo(dT) magnetic beads. Fragmentation reagents were added to the mRNA obtained in the previous step. A single-stranded synthesis reaction system was prepared, and the reaction programme was set to synthesize single-stranded cDNA; a double-stranded synthesis reaction system was prepared, and the reaction programme was set to synthesize double-stranded cDNA. The reaction system was prepared, and the reaction programme was set to repair the ends of the double-stranded cDNA, and an A base was added at the 3' end. The adapter ligation reaction system was prepared, and the reaction programme was set to connect the adapter with the cDNA. The PCR reaction system was prepared, and the reaction programme was set to amplify the product. The PCR products were denatured into a single strand, the circularization reaction system was prepared, and the reaction programme was set to obtain a single-stranded circular product and the linear DNA molecules that were not circularized were digested. The single-stranded circular DNA molecule is replicated by rolling circle replication to form a DNA nanoball (DNB) containing multiple copies. The obtained DNBs are placed into a mesh pore on a high-density DNA nanochip and sequenced using the combined probe anchor polymerization technology (cPAS).