RNA Sequencing Analysis of HCI-H1703 Cells Expressing Wild-Type (wt) PPM1G versus PPM1G Knockdown (Sh-PPM1G)

We established NCI-H1703 cells with stable PPM1G knockdown using short hairpin RNA (shRNA) technology. Subsequently, we performed data acquisition and analysis of high-throughput RNA sequencing (RNA-seq) by comparing these PPM1G-knockdown cells with wild-type NCI-H1703 cells. NCI-H1703 cells with PPM1G knockdown were constructed using short hairpin RNA (shRNA) technology. Both sh-PPM1G and wild-type (WT) tumor cells were seeded into cell culture flasks. When cell confluency reached 80%-90%, the cells were treated with 0.25% trypsin-EDTA solution: an appropriate volume of trypsin was added to cover the cell layer, and the flasks were incubated in a cell culture incubator for 1-2 minutes. Once cells appeared rounded and began to detach under microscopic observation, serum-containing medium was added to terminate digestion. The cell suspension was transferred to centrifuge tubes and centrifuged at 1500 rpm for 5 minutes. After discarding the supernatant, cell pellets were collected, gently resuspended in pre-chilled PBS, and centrifuged again for washing, repeating this process 2-3 times to completely remove residual medium components. The cells were then frozen in liquid nitrogen and sent to Hangzhou Astrocyte Technology Co., Ltd. for subsequent RNA sequencing (RNA-seq) procedures and data acquisition.