MG63 osteosarcoma cells were cultured in an incubator at 37℃ with 5%CO2. The medium was H-DMEM cell medium containing 100IU/ml penicillin, 10% thermal inactivated fetal bovine serum and 100ug/ml streptomycin. The medium was changed every 48 hours. ① The culture medium of 6-well plate cells was sucked, washed twice with PBS, 1 ml TRIZOL was added to each well, shaken well, and left for 3 minutes. ② The TRIZOL cell lysate from each well was sucked into the RNA-free EP tube, 0.2 ml chloroform was added, and gently shaken and mixed. ③ Centrifugation was performed at 12000 rpm at 4℃ for 15 min. The supernatant was added to the RNA-free EP tube, isopropyl alcohol was added and left for 10min. ④ Centrifuge at 12000 rpm for 10 min at 4℃. The supernatant was discarded, washed with 1.0ml 75% ethanol (newly prepared with DEPC water) and centrifuged at 7500rpm at 4℃ for 5 min. Discard the supernatant and drain the liquid. Dry RNAfor 5-10 min, add 20-30 uL water without RNase, and dissolve total RNA in 55℃ water bath for 5 min. OD values at 260nm and 280nm were measured. Total RNA purity 260/280=1.8-2.0 was considered qualified. The library was constructed by two - step PCR amplification.All PCR products were mixed according to the equimolar ratio after qPCR quantification. barcode and sequencing connector were added by PCR amplification method. The second generation sequencing library of target gene was constructed.