gRNA-seq from Ishikawa after medroxyprogesterone treatment
Hormone therapy serves as a primary choice for fertility preservation and is also considered for advanced and recurrent cases with endometrial cancer. While a number of patients fail to respond favorably to hormone treatments. In order to explore the mechanisms underpinning hormone resistance, Here we constructed a whole-genome library for EAC cell lines using the CRISPR-Cas9 system and performed high-throughput sequencing after medroxyprogesterone treatment in this study. For CRISPR screening, ishikawa cells were infected pooled GeCkov2 lentiviral library with functional MOI of 0.3. Genomic DNA from each group was isolated and amplified by PCR. The PCR products were purified and subjected to NGS by using the Novaseq 6000-PE150 platform. After 48h of puromycin selection, the cells were divided into three groups, the baseline group cells were collected and frozen at -80 degree centigrade, the control group cells were treated with DMSO for 10 days while the MPA group cells were treated with 15 µM MPA for 10 days. Overall design: Genome-wild lentiviral Human_GeCKOv2_Library _B was purchased from AZENTA LIFE SCIENCES . For CRISPR screening, 1×10^8 ishikawa cells were infected with pooled GeCKOv2 lentiviral library with functional MOI of 0.3. After 48h of puromycin selection, the cells were divided into three groups, the "baseline" group cells were collected and frozen at -80C, the "control" group cells were treated with DMSO for 10 days while the "MPA" group cells were treated with 15 µM MPA for 10 days. Genomic DNA from each group was isolated using HMV DNA Kit and amplified by PCR. The PCR products were purified and subjected to NGS by using the Novaseq 6000-PE150 platform.