title: GSM8016801: Screening_second; Homo sapiens; OTHER
library_construction_protocol: For our CRISPR screen, we first constructed a RAG library containing single guide RNAs (sgRNAs) targeting ribosome-related genes and based on the lentiCRISPRv2 plasmid (Addgene # 52961) as previously described. Then, we obtained a lentivirus library and infected senescent hMPCs at a low multiplicity of infection (MOI) (0.3) to ensure that one cell receives no more than one sgRNA. At the same time, we set up a control in which hMPCs were transduced with non-targeting control (NTC) at the same MOI. After two days, the cells transduced with sgRNA were put under puromycin selection (1 μg/ml) (InvivoGen) for six days, after which the cells switched to fresh medium without puromycin and passaged until the control cells underwent growth arrest. We harvested cells at different time points and extracted the genomic DNA using TIANamp Genomic DNA Kit (TIANGEN), according to the manufacturer's protocol. Finally, the sgRNA library was constructed as previously described (PMID: 33408182).