Generation of USP11 Knockout RPE1 Cells

USP11 KO RPE1 cells were generated using the CRISPR/Cas9 technology. Guide RNA sequences targeting spCas9 to the genomic locus of USP11 (Ensembl ID: ENSG00000102226) were designed according to (33 (link)). Specific overhangs for subsequent ligation into pLentiCRISPRv2 (gift from Feng Zhang, Addgene, plasmid #52961) were added to each guide (underlined):
USP11_KO-1-F: CACCGggtctccatgatgatcaact
USP11_KO-1-R: AAACagttgatcatcatggagaccCUSP11_KO-2-F: CACCGgtgggcgagaacgtccactg
USP11_KO-2-R: AAACcagtggacgttctcgcccacCUSP11_KO-3-F: CACCGtgataggcagtggaacactg
USP11_KO-3-R: AAACcagtgttccactgcctatcaCComplementary oligonucleotides were annealed for 5 min at 95 °C and subsequently cooled down for 15 min at room temperature (RT). Annealed primers were diluted to 0.5 μM in nuclease-free water and cloned into pLentiCRISPRv2 via BsmBI restriction enzyme (NEB) digest and subsequent ligation with T4 DNA ligase (NEB). Stellar competent cells (Clontech) were transformed with the ligation reaction, and correct clones were identified by Sanger sequencing (Microsynth Seqlab) using the U6 primer.