other:Article Title: Whole-genome CRISPR screening identifies genetic manipulations to reduce immune rejection of stem cell-derived islets
Article Snippet: Whole genome CRISPR screen in vivo and analysis Brunello pooled library pooled plasmid DNA in a 1 vector system (lentiCRISPRv2 backbone; Addgene # 73179) was obtained from the Broad institute Genetic Perturbation Platform (GPP), to generate pooled lentivirus.
Article Title: Whole-genome CRISPR screening identifies genetic manipulations to reduce immune rejection of stem cell-derived islets
Article Snippet: Whole genome CRISPR screen in vivo and analysis Brunello pooled library pooled plasmid DNA in a 1 vector system (lentiCRISPRv2 backbone; Addgene # 73179) was obtained from the Broad institute Genetic Perturbation Platform (GPP), to generate pooled lentivirus.
Construct:Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients
Article Snippet: .. To construct the lentiviral CRISPR–Cas9 vectors containing the desired sgRNA, the lentiCRISPRv2 backbone (Addgene, 52961) was digested with BsmBI. sgRNA oligos, which included CACC or AAAC overhangs, were synthesized by IDT, phosphorylated, annealed and ligated into the linearized backbone using T4 ligase (NEB, cat. no. M0202S). sgRNA DMXL1 GCTAGCAAAGAACCATCCAT sgRNA DMXL2 GTGACATTCTATAAAGATTG sgRNA WDR7 GGCAGCATTAGACCATGAAG dTAG-3xHA-P2A-Puro sequence: GGAGGCGGTTCAGGGGTACAGGTCGAGACAATAAGTCCGGGGGACGGTAGAACTTTCCCCAAAAGGGGCCAGACGTGTGTTGTGCATTATACGGGCATGTTGGAGGATGGAAAGAAAGTAGACTCCTCACGGGATAGAAACAAACCCTTTAAATTTATGTTGGGGAAGCAAGAGGTTATTAGGGGATGGGAAGAGGGTGTAGCCCAGATGTCTGTAGGCCAACGCGCAAAATTGACTATAAGTCCAGATTATGCGTACGGCGCTACCGGTCACCCTGGGATAATTCCCCCACACGCCACGCTGGTGTTTGATGTCGAACTTCTCAAACTGGAAGGCTCTGGTTATCCGTACGATGTACCTGATTATGCCGGGTATCCCTACGACGTGCCAGACTATGCTGGTTATCCGTATGACGTTCCGGACTATGCTGGTGCGACCAATTTTAGTTTGTTGAAACAGGCGGGTGATGTGGAGGAAAACCCGGGGCCTATGACCGAGTATAAACCCACCGTAAGACTGGCGACCAGGGACGACGTTCCAAGGGCTGTCCGGACGCTGGCTGCTGCGTTCGCGGATTACCCTGCCACTAGGCACACGGTTGACCCCGATCGACATATTGAAAGGGTAACTGAACTTCAAGAGTTGTTCCTGACGCGGGTAGGTCTGGATATAGGAAAAGTATGGGTAGCTGATGATGGCGCGGCTGTGGCAGTCTGGACCACTCCGGAAAGCGTTGAAGCTGGCGCAGTTTTCGCTGAAATCGGTCCCAGGATGGCAGAACTTAGTGGGTCCCGATTGGCGGCACAACAGCAAATGGAGGGACTGCTTGCACCGCACCGGCCTAAAGAGCCCGCGTGGTTCCTTGCCACCGTAGGTGTATCTCCTGATCACCAGGGAAAAGGTCTTGGCTCCGCCGTTGTGCTCCCGGGTGTTGAGGCTGCCGAAAGAGCAGGAGTCCCTGCATTTTTGGAAACTAGTGCCCCGCGAAACCTCCCTTTTTACGAACGCTTGGGTTTCACAGTCACGGCCGACGTTGAAGTCCCAGAGGGACCCAGAACGTGGTGCATGACCAGGAAACCCGGGGCT Helical bundle domain amino acid sequence: MYPYDVPDYAGGGSGQEHARVLSSHLMHSSLPGLTRLEQMFLVALADTVATTSTELDESRDKSCSGRDTLDECGLRYLLAMRLHTCLLTSLPPLYRVQLLHQGVSTCHFAWAFHSEAEEELINMIPAIQRGDPQWSELRAMGIGWWVRNINTLRRCIEKVAKASFQRNNDALDAALFYLSMKKKAVVWGLFRSQHDEKMTTFFSHNFNEDRWRKAALKNAFSLLGKQRFEQSAAFFLLAGSLKDAIEVCLEKMEDIQLAMVIARLYESEFETSSTYISILNQKILGCQKDGSGFSCKRLHPDPFLRSLAYWVMKDYTRALDTLLEQTPKEDDEHQVIIKSCNPVAFSFYNYLRTHPLLIRRNLASPEGTLATLGLKTEKNFVDKINLIERKLFFTTANAHFKVGCPVLALEVLSKI HA-DMXL2 WT or mutants were PCR amplified from the pRK5 vector. ..
Article Title: Radiation-induced nuclear translocation of NPRL2 hijacks E3 ubiquitin ligases to enhance DNA repair via the AMPK/WDR24 axis, contributing to CRC radioresistance
Article Snippet: .. The CRISPR-KO cell lines were constructed by cloning single-guide RNAs (sgRNAs) targeting NPRL2 (sg: 5′-GCAGAGGCTGGCCATACCAA-3′), DEPDC5 (sg: 5′-AAGGATCAGTATATTGGCCG-3′), WDR59 (sg: 5′-GGGACATTGGAGCTGTGCAG-3′), SEC13 (sg: 5′-GGTGTCAGTAATTAACACTG-3′), WDR24 (5′-GCACAGCTCAGGTTAAGCGA-3′), PRKAA1 (the gene encoding AMPK α 1, sg: 5′-GCAACTATCGATCTTGCCAA-3′), PRKAA2 (the gene encoding AMPK α 2, sg: 5′-GAAGAGTTGAGTACGCGCTC-3′) and Negative Control (Scrambled, sg: 5′-CGAGCTTGACTACTTGGCAA-3′) into the lentiCRISPRv2 backbone (Addgene [Watertown, MA, USA] plasmid No. 52961). ..
Article Title: Radiation-induced nuclear translocation of NPRL2 hijacks E3 ubiquitin ligases to enhance DNA repair via the AMPK/WDR24 axis, contributing to CRC radioresistance
Article Snippet: .. The CRISPR-KO cell lines were constructed by cloning singleguide RNAs (sgRNAs) targeting NPRL2 (sg: 5 ′ -GCAGAGGCTGGCCATACCAA-3 ′ ), DEPDC5 (sg: 5 ′ -AAGGATCAGTATATTGGCCG-3 ′ ), WDR59 (sg: 5 ′ -GGGACATTGGAGC TGTGCAG-3 ′ ), SEC13 (sg: 5 ′ -GGTGTCAGTAATTAACACTG3 ′ ), WDR24 (5 ′ -GCACAGCTCAGGTTAAGCGA-3 ′ ), PRKAA1 (the gene encoding AMPKα1, sg: 5 ′ -GCAACTATCGATCTTG CCAA-3 ′ ), PRKAA2 (the gene encoding AMPKα2, sg: 5 ′ -GAAGAGTTGAGTACGCGCTC-3 ′ ) and Negative Control (Scrambled, sg: 5 ′ -CGAGCTTGACTACTTGGCAA-3 ′ ) into the lentiCRISPRv2 backbone (Addgene [Watertown, MA, USA] plasmid No. 52961). ..
Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients.
Article Snippet: .. To construct the lentiviral CRISPR–Cas9 vectors containing the desired sgRNA, the lentiCRISPRv2 backbone (Addgene, 52961) was digested with BsmBI. sgRNA oligos, which included CACC or AAAC overhangs, were synthesized by IDT, phosphorylated, annealed and ligated into the linearized backbone using T4 ligase (NEB, cat. no. M0202S). sgRNA DMXL1 GCTAGCAAAGAACCATCCAT sgRNA DMXL2 GTGACATTCTATAAAGATTG sgRNA WDR7 GGCAGCATTAGACCATGAAG dTAG-3xHA-P2A-Puro sequence: GGAGGCGGTTCAGGGGTACAGGTCGAGACAATAAGTCCGGGGGACGGTAGAACTTTCCCCAAAAGGGGCCAGACGTGTGTTGTGCATTATACGGGCATGTTGGAGGATGGAAAGAAAGTAGACTCCTCACGGGATAGAAACAAACCCTTTAAATTTATGTTGGGGAAGCAAGAGGTTATTAGGGGATGGGAAGAGGGTGTAGCCCAGATGTCTGTAGGCCAACGCGCAAAATTGACTATAAGTCCAGATTATGCGTACGGCGCTACCGGTCACCCTGGGATAATTCCCCCACACGCCACGCTGGTGTTTGATGTCGAACTTCTCAAACTGGAAGGCTCTGGTTATCCGTACGATGTAC- CTGATTATGCCGGGTATCCCTACGACGTGCCAGACTATGCTGGTTATCCGTATGACGTTCCGGACTATGCTGGTGCGACCAATTTTAGTTTGTTGAAACAGGCGGGTGATGTGGAGGAAAACCCGGGGCCTATGACCGAGTATAAACCCACCGTAAGACTGGCGACCAGGGACGACGTTCCAAGGGCTGTCCGGACGCTGGCTGCTGCGTTCGCGGATTACCCTGCCACTAGGCACACGGTTGACCCCGATCGACATATTGAAAGGGTAACTGAACTTCAAGAGTTGTTCCTGACGCGGGTAGGTCTGGATATAGGAAAAGTATGGGTAGCTGATGATGGCGCGGCTGTGGCAGTCTGGACCACTCCGGAAAGCGTTGAAGCTGGCGCAGTTTTCGCTGAAATCGGTCCCAGGATGGCAGAACTTAGTGGGTCCCGATTGGCGGCACAACAGCAAATGGAGGGACTGCTTGCACCGCACCGGCCTAAAGAGCCCGCGTGGTTCCTTGCCACCGTAGGTGTATCTCCTGATCACCAGGGAAAAGGTCTTGGCTCCGCCGTTGTGCTCCCGGGTGTTGAGGCTGCCGAAAGAGCAGGAGTCCCTGCATTTTTGGAAACTAGTGCCCCGCGAAACCTCCCTTTTTACGAACGCTTGGGTTTCACAGTCACGGCCGACGTTGAAGTCCCAGAGGGACCCAGAACGTGGTGCATGACCAGGAAACCCGGGGCT Helical bundle domain amino acid sequence: MYPYDVPDYAGGGSGQEHARVLSSHLMHSSLPGLTRLEQMFLVALADTVATTSTELDESRDKSCSGRDTLDECGLRYLLAMRLHTCLLTSLPPLYRVQLLHQGVSTCHFAWAFHSEAEEELINMIPAIQRGDPQWSELRAMGIGWWVRNINTLRRCIEKVAKASFQRNNDALDAALFYLSMKKKAVVWGLFRSQHDEKMTTFFSHNFNEDRWRKAALKNAFSLLGKQRFEQSAAFFLLAGSLKDAIEVCLEKMEDIQLAMVIARLYESEFETSSTYISILNQKILGCQKDGSGFSCKRLHPDPFLRSLAYWVMKDYTRALDTLLEQTPKEDDEHQVIIKSCNPVAFSFYNYLRTHPLLIRRNLASPEGTLATLGLKTEKNFVDKINLIERKLFFTTANAHFKVGCPVLALEVLSKI HA-DMXL2 WT or mutants were PCR amplified from the pRK5 vector. ..
CRISPR:Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients
Article Snippet: .. To construct the lentiviral CRISPR–Cas9 vectors containing the desired sgRNA, the lentiCRISPRv2 backbone (Addgene, 52961) was digested with BsmBI. sgRNA oligos, which included CACC or AAAC overhangs, were synthesized by IDT, phosphorylated, annealed and ligated into the linearized backbone using T4 ligase (NEB, cat. no. M0202S). sgRNA DMXL1 GCTAGCAAAGAACCATCCAT sgRNA DMXL2 GTGACATTCTATAAAGATTG sgRNA WDR7 GGCAGCATTAGACCATGAAG dTAG-3xHA-P2A-Puro sequence: GGAGGCGGTTCAGGGGTACAGGTCGAGACAATAAGTCCGGGGGACGGTAGAACTTTCCCCAAAAGGGGCCAGACGTGTGTTGTGCATTATACGGGCATGTTGGAGGATGGAAAGAAAGTAGACTCCTCACGGGATAGAAACAAACCCTTTAAATTTATGTTGGGGAAGCAAGAGGTTATTAGGGGATGGGAAGAGGGTGTAGCCCAGATGTCTGTAGGCCAACGCGCAAAATTGACTATAAGTCCAGATTATGCGTACGGCGCTACCGGTCACCCTGGGATAATTCCCCCACACGCCACGCTGGTGTTTGATGTCGAACTTCTCAAACTGGAAGGCTCTGGTTATCCGTACGATGTACCTGATTATGCCGGGTATCCCTACGACGTGCCAGACTATGCTGGTTATCCGTATGACGTTCCGGACTATGCTGGTGCGACCAATTTTAGTTTGTTGAAACAGGCGGGTGATGTGGAGGAAAACCCGGGGCCTATGACCGAGTATAAACCCACCGTAAGACTGGCGACCAGGGACGACGTTCCAAGGGCTGTCCGGACGCTGGCTGCTGCGTTCGCGGATTACCCTGCCACTAGGCACACGGTTGACCCCGATCGACATATTGAAAGGGTAACTGAACTTCAAGAGTTGTTCCTGACGCGGGTAGGTCTGGATATAGGAAAAGTATGGGTAGCTGATGATGGCGCGGCTGTGGCAGTCTGGACCACTCCGGAAAGCGTTGAAGCTGGCGCAGTTTTCGCTGAAATCGGTCCCAGGATGGCAGAACTTAGTGGGTCCCGATTGGCGGCACAACAGCAAATGGAGGGACTGCTTGCACCGCACCGGCCTAAAGAGCCCGCGTGGTTCCTTGCCACCGTAGGTGTATCTCCTGATCACCAGGGAAAAGGTCTTGGCTCCGCCGTTGTGCTCCCGGGTGTTGAGGCTGCCGAAAGAGCAGGAGTCCCTGCATTTTTGGAAACTAGTGCCCCGCGAAACCTCCCTTTTTACGAACGCTTGGGTTTCACAGTCACGGCCGACGTTGAAGTCCCAGAGGGACCCAGAACGTGGTGCATGACCAGGAAACCCGGGGCT Helical bundle domain amino acid sequence: MYPYDVPDYAGGGSGQEHARVLSSHLMHSSLPGLTRLEQMFLVALADTVATTSTELDESRDKSCSGRDTLDECGLRYLLAMRLHTCLLTSLPPLYRVQLLHQGVSTCHFAWAFHSEAEEELINMIPAIQRGDPQWSELRAMGIGWWVRNINTLRRCIEKVAKASFQRNNDALDAALFYLSMKKKAVVWGLFRSQHDEKMTTFFSHNFNEDRWRKAALKNAFSLLGKQRFEQSAAFFLLAGSLKDAIEVCLEKMEDIQLAMVIARLYESEFETSSTYISILNQKILGCQKDGSGFSCKRLHPDPFLRSLAYWVMKDYTRALDTLLEQTPKEDDEHQVIIKSCNPVAFSFYNYLRTHPLLIRRNLASPEGTLATLGLKTEKNFVDKINLIERKLFFTTANAHFKVGCPVLALEVLSKI HA-DMXL2 WT or mutants were PCR amplified from the pRK5 vector. ..
Article Title: Radiation-induced nuclear translocation of NPRL2 hijacks E3 ubiquitin ligases to enhance DNA repair via the AMPK/WDR24 axis, contributing to CRC radioresistance
Article Snippet: .. The CRISPR-KO cell lines were constructed by cloning single-guide RNAs (sgRNAs) targeting NPRL2 (sg: 5′-GCAGAGGCTGGCCATACCAA-3′), DEPDC5 (sg: 5′-AAGGATCAGTATATTGGCCG-3′), WDR59 (sg: 5′-GGGACATTGGAGCTGTGCAG-3′), SEC13 (sg: 5′-GGTGTCAGTAATTAACACTG-3′), WDR24 (5′-GCACAGCTCAGGTTAAGCGA-3′), PRKAA1 (the gene encoding AMPK α 1, sg: 5′-GCAACTATCGATCTTGCCAA-3′), PRKAA2 (the gene encoding AMPK α 2, sg: 5′-GAAGAGTTGAGTACGCGCTC-3′) and Negative Control (Scrambled, sg: 5′-CGAGCTTGACTACTTGGCAA-3′) into the lentiCRISPRv2 backbone (Addgene [Watertown, MA, USA] plasmid No. 52961). ..
Article Title: Radiation-induced nuclear translocation of NPRL2 hijacks E3 ubiquitin ligases to enhance DNA repair via the AMPK/WDR24 axis, contributing to CRC radioresistance
Article Snippet: .. The CRISPR-KO cell lines were constructed by cloning singleguide RNAs (sgRNAs) targeting NPRL2 (sg: 5 ′ -GCAGAGGCTGGCCATACCAA-3 ′ ), DEPDC5 (sg: 5 ′ -AAGGATCAGTATATTGGCCG-3 ′ ), WDR59 (sg: 5 ′ -GGGACATTGGAGC TGTGCAG-3 ′ ), SEC13 (sg: 5 ′ -GGTGTCAGTAATTAACACTG3 ′ ), WDR24 (5 ′ -GCACAGCTCAGGTTAAGCGA-3 ′ ), PRKAA1 (the gene encoding AMPKα1, sg: 5 ′ -GCAACTATCGATCTTG CCAA-3 ′ ), PRKAA2 (the gene encoding AMPKα2, sg: 5 ′ -GAAGAGTTGAGTACGCGCTC-3 ′ ) and Negative Control (Scrambled, sg: 5 ′ -CGAGCTTGACTACTTGGCAA-3 ′ ) into the lentiCRISPRv2 backbone (Addgene [Watertown, MA, USA] plasmid No. 52961). ..
Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients.
Article Snippet: .. To construct the lentiviral CRISPR–Cas9 vectors containing the desired sgRNA, the lentiCRISPRv2 backbone (Addgene, 52961) was digested with BsmBI. sgRNA oligos, which included CACC or AAAC overhangs, were synthesized by IDT, phosphorylated, annealed and ligated into the linearized backbone using T4 ligase (NEB, cat. no. M0202S). sgRNA DMXL1 GCTAGCAAAGAACCATCCAT sgRNA DMXL2 GTGACATTCTATAAAGATTG sgRNA WDR7 GGCAGCATTAGACCATGAAG dTAG-3xHA-P2A-Puro sequence: GGAGGCGGTTCAGGGGTACAGGTCGAGACAATAAGTCCGGGGGACGGTAGAACTTTCCCCAAAAGGGGCCAGACGTGTGTTGTGCATTATACGGGCATGTTGGAGGATGGAAAGAAAGTAGACTCCTCACGGGATAGAAACAAACCCTTTAAATTTATGTTGGGGAAGCAAGAGGTTATTAGGGGATGGGAAGAGGGTGTAGCCCAGATGTCTGTAGGCCAACGCGCAAAATTGACTATAAGTCCAGATTATGCGTACGGCGCTACCGGTCACCCTGGGATAATTCCCCCACACGCCACGCTGGTGTTTGATGTCGAACTTCTCAAACTGGAAGGCTCTGGTTATCCGTACGATGTAC- CTGATTATGCCGGGTATCCCTACGACGTGCCAGACTATGCTGGTTATCCGTATGACGTTCCGGACTATGCTGGTGCGACCAATTTTAGTTTGTTGAAACAGGCGGGTGATGTGGAGGAAAACCCGGGGCCTATGACCGAGTATAAACCCACCGTAAGACTGGCGACCAGGGACGACGTTCCAAGGGCTGTCCGGACGCTGGCTGCTGCGTTCGCGGATTACCCTGCCACTAGGCACACGGTTGACCCCGATCGACATATTGAAAGGGTAACTGAACTTCAAGAGTTGTTCCTGACGCGGGTAGGTCTGGATATAGGAAAAGTATGGGTAGCTGATGATGGCGCGGCTGTGGCAGTCTGGACCACTCCGGAAAGCGTTGAAGCTGGCGCAGTTTTCGCTGAAATCGGTCCCAGGATGGCAGAACTTAGTGGGTCCCGATTGGCGGCACAACAGCAAATGGAGGGACTGCTTGCACCGCACCGGCCTAAAGAGCCCGCGTGGTTCCTTGCCACCGTAGGTGTATCTCCTGATCACCAGGGAAAAGGTCTTGGCTCCGCCGTTGTGCTCCCGGGTGTTGAGGCTGCCGAAAGAGCAGGAGTCCCTGCATTTTTGGAAACTAGTGCCCCGCGAAACCTCCCTTTTTACGAACGCTTGGGTTTCACAGTCACGGCCGACGTTGAAGTCCCAGAGGGACCCAGAACGTGGTGCATGACCAGGAAACCCGGGGCT Helical bundle domain amino acid sequence: MYPYDVPDYAGGGSGQEHARVLSSHLMHSSLPGLTRLEQMFLVALADTVATTSTELDESRDKSCSGRDTLDECGLRYLLAMRLHTCLLTSLPPLYRVQLLHQGVSTCHFAWAFHSEAEEELINMIPAIQRGDPQWSELRAMGIGWWVRNINTLRRCIEKVAKASFQRNNDALDAALFYLSMKKKAVVWGLFRSQHDEKMTTFFSHNFNEDRWRKAALKNAFSLLGKQRFEQSAAFFLLAGSLKDAIEVCLEKMEDIQLAMVIARLYESEFETSSTYISILNQKILGCQKDGSGFSCKRLHPDPFLRSLAYWVMKDYTRALDTLLEQTPKEDDEHQVIIKSCNPVAFSFYNYLRTHPLLIRRNLASPEGTLATLGLKTEKNFVDKINLIERKLFFTTANAHFKVGCPVLALEVLSKI HA-DMXL2 WT or mutants were PCR amplified from the pRK5 vector. ..
Article Title: Cell heterogeneity and fate bistability drive tissue patterning during intestinal regeneration
Article Snippet: After that maintenance of organoid cultures was performed in IntestiCult Organoid Growth Medium (STEMCELL Technologies) with 100μg/ml Penicillin-Streptomycin. .. For the CRISPR-Cas9 mediated knockout of FOXA1, 20bp long guide sequences were designed using the online CHOPCHOP tool ( https://chopchop.cbu.uib.no ) ( ) and cloned into the lentiCRISPRv2 backbone (Addgene plasmid #52961) using the protocol described previously ( ). .. Briefly, vectors were digested using BsmBI and used for ligation with the phosphorylated sgRNA oligos.
Synthesized:Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients
Article Snippet: .. To construct the lentiviral CRISPR–Cas9 vectors containing the desired sgRNA, the lentiCRISPRv2 backbone (Addgene, 52961) was digested with BsmBI. sgRNA oligos, which included CACC or AAAC overhangs, were synthesized by IDT, phosphorylated, annealed and ligated into the linearized backbone using T4 ligase (NEB, cat. no. M0202S). sgRNA DMXL1 GCTAGCAAAGAACCATCCAT sgRNA DMXL2 GTGACATTCTATAAAGATTG sgRNA WDR7 GGCAGCATTAGACCATGAAG dTAG-3xHA-P2A-Puro sequence: GGAGGCGGTTCAGGGGTACAGGTCGAGACAATAAGTCCGGGGGACGGTAGAACTTTCCCCAAAAGGGGCCAGACGTGTGTTGTGCATTATACGGGCATGTTGGAGGATGGAAAGAAAGTAGACTCCTCACGGGATAGAAACAAACCCTTTAAATTTATGTTGGGGAAGCAAGAGGTTATTAGGGGATGGGAAGAGGGTGTAGCCCAGATGTCTGTAGGCCAACGCGCAAAATTGACTATAAGTCCAGATTATGCGTACGGCGCTACCGGTCACCCTGGGATAATTCCCCCACACGCCACGCTGGTGTTTGATGTCGAACTTCTCAAACTGGAAGGCTCTGGTTATCCGTACGATGTACCTGATTATGCCGGGTATCCCTACGACGTGCCAGACTATGCTGGTTATCCGTATGACGTTCCGGACTATGCTGGTGCGACCAATTTTAGTTTGTTGAAACAGGCGGGTGATGTGGAGGAAAACCCGGGGCCTATGACCGAGTATAAACCCACCGTAAGACTGGCGACCAGGGACGACGTTCCAAGGGCTGTCCGGACGCTGGCTGCTGCGTTCGCGGATTACCCTGCCACTAGGCACACGGTTGACCCCGATCGACATATTGAAAGGGTAACTGAACTTCAAGAGTTGTTCCTGACGCGGGTAGGTCTGGATATAGGAAAAGTATGGGTAGCTGATGATGGCGCGGCTGTGGCAGTCTGGACCACTCCGGAAAGCGTTGAAGCTGGCGCAGTTTTCGCTGAAATCGGTCCCAGGATGGCAGAACTTAGTGGGTCCCGATTGGCGGCACAACAGCAAATGGAGGGACTGCTTGCACCGCACCGGCCTAAAGAGCCCGCGTGGTTCCTTGCCACCGTAGGTGTATCTCCTGATCACCAGGGAAAAGGTCTTGGCTCCGCCGTTGTGCTCCCGGGTGTTGAGGCTGCCGAAAGAGCAGGAGTCCCTGCATTTTTGGAAACTAGTGCCCCGCGAAACCTCCCTTTTTACGAACGCTTGGGTTTCACAGTCACGGCCGACGTTGAAGTCCCAGAGGGACCCAGAACGTGGTGCATGACCAGGAAACCCGGGGCT Helical bundle domain amino acid sequence: MYPYDVPDYAGGGSGQEHARVLSSHLMHSSLPGLTRLEQMFLVALADTVATTSTELDESRDKSCSGRDTLDECGLRYLLAMRLHTCLLTSLPPLYRVQLLHQGVSTCHFAWAFHSEAEEELINMIPAIQRGDPQWSELRAMGIGWWVRNINTLRRCIEKVAKASFQRNNDALDAALFYLSMKKKAVVWGLFRSQHDEKMTTFFSHNFNEDRWRKAALKNAFSLLGKQRFEQSAAFFLLAGSLKDAIEVCLEKMEDIQLAMVIARLYESEFETSSTYISILNQKILGCQKDGSGFSCKRLHPDPFLRSLAYWVMKDYTRALDTLLEQTPKEDDEHQVIIKSCNPVAFSFYNYLRTHPLLIRRNLASPEGTLATLGLKTEKNFVDKINLIERKLFFTTANAHFKVGCPVLALEVLSKI HA-DMXL2 WT or mutants were PCR amplified from the pRK5 vector. ..
Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients.
Article Snippet: .. To construct the lentiviral CRISPR–Cas9 vectors containing the desired sgRNA, the lentiCRISPRv2 backbone (Addgene, 52961) was digested with BsmBI. sgRNA oligos, which included CACC or AAAC overhangs, were synthesized by IDT, phosphorylated, annealed and ligated into the linearized backbone using T4 ligase (NEB, cat. no. M0202S). sgRNA DMXL1 GCTAGCAAAGAACCATCCAT sgRNA DMXL2 GTGACATTCTATAAAGATTG sgRNA WDR7 GGCAGCATTAGACCATGAAG dTAG-3xHA-P2A-Puro sequence: GGAGGCGGTTCAGGGGTACAGGTCGAGACAATAAGTCCGGGGGACGGTAGAACTTTCCCCAAAAGGGGCCAGACGTGTGTTGTGCATTATACGGGCATGTTGGAGGATGGAAAGAAAGTAGACTCCTCACGGGATAGAAACAAACCCTTTAAATTTATGTTGGGGAAGCAAGAGGTTATTAGGGGATGGGAAGAGGGTGTAGCCCAGATGTCTGTAGGCCAACGCGCAAAATTGACTATAAGTCCAGATTATGCGTACGGCGCTACCGGTCACCCTGGGATAATTCCCCCACACGCCACGCTGGTGTTTGATGTCGAACTTCTCAAACTGGAAGGCTCTGGTTATCCGTACGATGTAC- CTGATTATGCCGGGTATCCCTACGACGTGCCAGACTATGCTGGTTATCCGTATGACGTTCCGGACTATGCTGGTGCGACCAATTTTAGTTTGTTGAAACAGGCGGGTGATGTGGAGGAAAACCCGGGGCCTATGACCGAGTATAAACCCACCGTAAGACTGGCGACCAGGGACGACGTTCCAAGGGCTGTCCGGACGCTGGCTGCTGCGTTCGCGGATTACCCTGCCACTAGGCACACGGTTGACCCCGATCGACATATTGAAAGGGTAACTGAACTTCAAGAGTTGTTCCTGACGCGGGTAGGTCTGGATATAGGAAAAGTATGGGTAGCTGATGATGGCGCGGCTGTGGCAGTCTGGACCACTCCGGAAAGCGTTGAAGCTGGCGCAGTTTTCGCTGAAATCGGTCCCAGGATGGCAGAACTTAGTGGGTCCCGATTGGCGGCACAACAGCAAATGGAGGGACTGCTTGCACCGCACCGGCCTAAAGAGCCCGCGTGGTTCCTTGCCACCGTAGGTGTATCTCCTGATCACCAGGGAAAAGGTCTTGGCTCCGCCGTTGTGCTCCCGGGTGTTGAGGCTGCCGAAAGAGCAGGAGTCCCTGCATTTTTGGAAACTAGTGCCCCGCGAAACCTCCCTTTTTACGAACGCTTGGGTTTCACAGTCACGGCCGACGTTGAAGTCCCAGAGGGACCCAGAACGTGGTGCATGACCAGGAAACCCGGGGCT Helical bundle domain amino acid sequence: MYPYDVPDYAGGGSGQEHARVLSSHLMHSSLPGLTRLEQMFLVALADTVATTSTELDESRDKSCSGRDTLDECGLRYLLAMRLHTCLLTSLPPLYRVQLLHQGVSTCHFAWAFHSEAEEELINMIPAIQRGDPQWSELRAMGIGWWVRNINTLRRCIEKVAKASFQRNNDALDAALFYLSMKKKAVVWGLFRSQHDEKMTTFFSHNFNEDRWRKAALKNAFSLLGKQRFEQSAAFFLLAGSLKDAIEVCLEKMEDIQLAMVIARLYESEFETSSTYISILNQKILGCQKDGSGFSCKRLHPDPFLRSLAYWVMKDYTRALDTLLEQTPKEDDEHQVIIKSCNPVAFSFYNYLRTHPLLIRRNLASPEGTLATLGLKTEKNFVDKINLIERKLFFTTANAHFKVGCPVLALEVLSKI HA-DMXL2 WT or mutants were PCR amplified from the pRK5 vector. ..
Sequencing:Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients
Article Snippet: .. To construct the lentiviral CRISPR–Cas9 vectors containing the desired sgRNA, the lentiCRISPRv2 backbone (Addgene, 52961) was digested with BsmBI. sgRNA oligos, which included CACC or AAAC overhangs, were synthesized by IDT, phosphorylated, annealed and ligated into the linearized backbone using T4 ligase (NEB, cat. no. M0202S). sgRNA DMXL1 GCTAGCAAAGAACCATCCAT sgRNA DMXL2 GTGACATTCTATAAAGATTG sgRNA WDR7 GGCAGCATTAGACCATGAAG dTAG-3xHA-P2A-Puro sequence: GGAGGCGGTTCAGGGGTACAGGTCGAGACAATAAGTCCGGGGGACGGTAGAACTTTCCCCAAAAGGGGCCAGACGTGTGTTGTGCATTATACGGGCATGTTGGAGGATGGAAAGAAAGTAGACTCCTCACGGGATAGAAACAAACCCTTTAAATTTATGTTGGGGAAGCAAGAGGTTATTAGGGGATGGGAAGAGGGTGTAGCCCAGATGTCTGTAGGCCAACGCGCAAAATTGACTATAAGTCCAGATTATGCGTACGGCGCTACCGGTCACCCTGGGATAATTCCCCCACACGCCACGCTGGTGTTTGATGTCGAACTTCTCAAACTGGAAGGCTCTGGTTATCCGTACGATGTACCTGATTATGCCGGGTATCCCTACGACGTGCCAGACTATGCTGGTTATCCGTATGACGTTCCGGACTATGCTGGTGCGACCAATTTTAGTTTGTTGAAACAGGCGGGTGATGTGGAGGAAAACCCGGGGCCTATGACCGAGTATAAACCCACCGTAAGACTGGCGACCAGGGACGACGTTCCAAGGGCTGTCCGGACGCTGGCTGCTGCGTTCGCGGATTACCCTGCCACTAGGCACACGGTTGACCCCGATCGACATATTGAAAGGGTAACTGAACTTCAAGAGTTGTTCCTGACGCGGGTAGGTCTGGATATAGGAAAAGTATGGGTAGCTGATGATGGCGCGGCTGTGGCAGTCTGGACCACTCCGGAAAGCGTTGAAGCTGGCGCAGTTTTCGCTGAAATCGGTCCCAGGATGGCAGAACTTAGTGGGTCCCGATTGGCGGCACAACAGCAAATGGAGGGACTGCTTGCACCGCACCGGCCTAAAGAGCCCGCGTGGTTCCTTGCCACCGTAGGTGTATCTCCTGATCACCAGGGAAAAGGTCTTGGCTCCGCCGTTGTGCTCCCGGGTGTTGAGGCTGCCGAAAGAGCAGGAGTCCCTGCATTTTTGGAAACTAGTGCCCCGCGAAACCTCCCTTTTTACGAACGCTTGGGTTTCACAGTCACGGCCGACGTTGAAGTCCCAGAGGGACCCAGAACGTGGTGCATGACCAGGAAACCCGGGGCT Helical bundle domain amino acid sequence: MYPYDVPDYAGGGSGQEHARVLSSHLMHSSLPGLTRLEQMFLVALADTVATTSTELDESRDKSCSGRDTLDECGLRYLLAMRLHTCLLTSLPPLYRVQLLHQGVSTCHFAWAFHSEAEEELINMIPAIQRGDPQWSELRAMGIGWWVRNINTLRRCIEKVAKASFQRNNDALDAALFYLSMKKKAVVWGLFRSQHDEKMTTFFSHNFNEDRWRKAALKNAFSLLGKQRFEQSAAFFLLAGSLKDAIEVCLEKMEDIQLAMVIARLYESEFETSSTYISILNQKILGCQKDGSGFSCKRLHPDPFLRSLAYWVMKDYTRALDTLLEQTPKEDDEHQVIIKSCNPVAFSFYNYLRTHPLLIRRNLASPEGTLATLGLKTEKNFVDKINLIERKLFFTTANAHFKVGCPVLALEVLSKI HA-DMXL2 WT or mutants were PCR amplified from the pRK5 vector. ..
Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients.
Article Snippet: .. To construct the lentiviral CRISPR–Cas9 vectors containing the desired sgRNA, the lentiCRISPRv2 backbone (Addgene, 52961) was digested with BsmBI. sgRNA oligos, which included CACC or AAAC overhangs, were synthesized by IDT, phosphorylated, annealed and ligated into the linearized backbone using T4 ligase (NEB, cat. no. M0202S). sgRNA DMXL1 GCTAGCAAAGAACCATCCAT sgRNA DMXL2 GTGACATTCTATAAAGATTG sgRNA WDR7 GGCAGCATTAGACCATGAAG dTAG-3xHA-P2A-Puro sequence: GGAGGCGGTTCAGGGGTACAGGTCGAGACAATAAGTCCGGGGGACGGTAGAACTTTCCCCAAAAGGGGCCAGACGTGTGTTGTGCATTATACGGGCATGTTGGAGGATGGAAAGAAAGTAGACTCCTCACGGGATAGAAACAAACCCTTTAAATTTATGTTGGGGAAGCAAGAGGTTATTAGGGGATGGGAAGAGGGTGTAGCCCAGATGTCTGTAGGCCAACGCGCAAAATTGACTATAAGTCCAGATTATGCGTACGGCGCTACCGGTCACCCTGGGATAATTCCCCCACACGCCACGCTGGTGTTTGATGTCGAACTTCTCAAACTGGAAGGCTCTGGTTATCCGTACGATGTAC- CTGATTATGCCGGGTATCCCTACGACGTGCCAGACTATGCTGGTTATCCGTATGACGTTCCGGACTATGCTGGTGCGACCAATTTTAGTTTGTTGAAACAGGCGGGTGATGTGGAGGAAAACCCGGGGCCTATGACCGAGTATAAACCCACCGTAAGACTGGCGACCAGGGACGACGTTCCAAGGGCTGTCCGGACGCTGGCTGCTGCGTTCGCGGATTACCCTGCCACTAGGCACACGGTTGACCCCGATCGACATATTGAAAGGGTAACTGAACTTCAAGAGTTGTTCCTGACGCGGGTAGGTCTGGATATAGGAAAAGTATGGGTAGCTGATGATGGCGCGGCTGTGGCAGTCTGGACCACTCCGGAAAGCGTTGAAGCTGGCGCAGTTTTCGCTGAAATCGGTCCCAGGATGGCAGAACTTAGTGGGTCCCGATTGGCGGCACAACAGCAAATGGAGGGACTGCTTGCACCGCACCGGCCTAAAGAGCCCGCGTGGTTCCTTGCCACCGTAGGTGTATCTCCTGATCACCAGGGAAAAGGTCTTGGCTCCGCCGTTGTGCTCCCGGGTGTTGAGGCTGCCGAAAGAGCAGGAGTCCCTGCATTTTTGGAAACTAGTGCCCCGCGAAACCTCCCTTTTTACGAACGCTTGGGTTTCACAGTCACGGCCGACGTTGAAGTCCCAGAGGGACCCAGAACGTGGTGCATGACCAGGAAACCCGGGGCT Helical bundle domain amino acid sequence: MYPYDVPDYAGGGSGQEHARVLSSHLMHSSLPGLTRLEQMFLVALADTVATTSTELDESRDKSCSGRDTLDECGLRYLLAMRLHTCLLTSLPPLYRVQLLHQGVSTCHFAWAFHSEAEEELINMIPAIQRGDPQWSELRAMGIGWWVRNINTLRRCIEKVAKASFQRNNDALDAALFYLSMKKKAVVWGLFRSQHDEKMTTFFSHNFNEDRWRKAALKNAFSLLGKQRFEQSAAFFLLAGSLKDAIEVCLEKMEDIQLAMVIARLYESEFETSSTYISILNQKILGCQKDGSGFSCKRLHPDPFLRSLAYWVMKDYTRALDTLLEQTPKEDDEHQVIIKSCNPVAFSFYNYLRTHPLLIRRNLASPEGTLATLGLKTEKNFVDKINLIERKLFFTTANAHFKVGCPVLALEVLSKI HA-DMXL2 WT or mutants were PCR amplified from the pRK5 vector. ..
Polymerase Chain Reaction:Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients
Article Snippet: .. To construct the lentiviral CRISPR–Cas9 vectors containing the desired sgRNA, the lentiCRISPRv2 backbone (Addgene, 52961) was digested with BsmBI. sgRNA oligos, which included CACC or AAAC overhangs, were synthesized by IDT, phosphorylated, annealed and ligated into the linearized backbone using T4 ligase (NEB, cat. no. M0202S). sgRNA DMXL1 GCTAGCAAAGAACCATCCAT sgRNA DMXL2 GTGACATTCTATAAAGATTG sgRNA WDR7 GGCAGCATTAGACCATGAAG dTAG-3xHA-P2A-Puro sequence: GGAGGCGGTTCAGGGGTACAGGTCGAGACAATAAGTCCGGGGGACGGTAGAACTTTCCCCAAAAGGGGCCAGACGTGTGTTGTGCATTATACGGGCATGTTGGAGGATGGAAAGAAAGTAGACTCCTCACGGGATAGAAACAAACCCTTTAAATTTATGTTGGGGAAGCAAGAGGTTATTAGGGGATGGGAAGAGGGTGTAGCCCAGATGTCTGTAGGCCAACGCGCAAAATTGACTATAAGTCCAGATTATGCGTACGGCGCTACCGGTCACCCTGGGATAATTCCCCCACACGCCACGCTGGTGTTTGATGTCGAACTTCTCAAACTGGAAGGCTCTGGTTATCCGTACGATGTACCTGATTATGCCGGGTATCCCTACGACGTGCCAGACTATGCTGGTTATCCGTATGACGTTCCGGACTATGCTGGTGCGACCAATTTTAGTTTGTTGAAACAGGCGGGTGATGTGGAGGAAAACCCGGGGCCTATGACCGAGTATAAACCCACCGTAAGACTGGCGACCAGGGACGACGTTCCAAGGGCTGTCCGGACGCTGGCTGCTGCGTTCGCGGATTACCCTGCCACTAGGCACACGGTTGACCCCGATCGACATATTGAAAGGGTAACTGAACTTCAAGAGTTGTTCCTGACGCGGGTAGGTCTGGATATAGGAAAAGTATGGGTAGCTGATGATGGCGCGGCTGTGGCAGTCTGGACCACTCCGGAAAGCGTTGAAGCTGGCGCAGTTTTCGCTGAAATCGGTCCCAGGATGGCAGAACTTAGTGGGTCCCGATTGGCGGCACAACAGCAAATGGAGGGACTGCTTGCACCGCACCGGCCTAAAGAGCCCGCGTGGTTCCTTGCCACCGTAGGTGTATCTCCTGATCACCAGGGAAAAGGTCTTGGCTCCGCCGTTGTGCTCCCGGGTGTTGAGGCTGCCGAAAGAGCAGGAGTCCCTGCATTTTTGGAAACTAGTGCCCCGCGAAACCTCCCTTTTTACGAACGCTTGGGTTTCACAGTCACGGCCGACGTTGAAGTCCCAGAGGGACCCAGAACGTGGTGCATGACCAGGAAACCCGGGGCT Helical bundle domain amino acid sequence: MYPYDVPDYAGGGSGQEHARVLSSHLMHSSLPGLTRLEQMFLVALADTVATTSTELDESRDKSCSGRDTLDECGLRYLLAMRLHTCLLTSLPPLYRVQLLHQGVSTCHFAWAFHSEAEEELINMIPAIQRGDPQWSELRAMGIGWWVRNINTLRRCIEKVAKASFQRNNDALDAALFYLSMKKKAVVWGLFRSQHDEKMTTFFSHNFNEDRWRKAALKNAFSLLGKQRFEQSAAFFLLAGSLKDAIEVCLEKMEDIQLAMVIARLYESEFETSSTYISILNQKILGCQKDGSGFSCKRLHPDPFLRSLAYWVMKDYTRALDTLLEQTPKEDDEHQVIIKSCNPVAFSFYNYLRTHPLLIRRNLASPEGTLATLGLKTEKNFVDKINLIERKLFFTTANAHFKVGCPVLALEVLSKI HA-DMXL2 WT or mutants were PCR amplified from the pRK5 vector. ..
Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients.
Article Snippet: .. To construct the lentiviral CRISPR–Cas9 vectors containing the desired sgRNA, the lentiCRISPRv2 backbone (Addgene, 52961) was digested with BsmBI. sgRNA oligos, which included CACC or AAAC overhangs, were synthesized by IDT, phosphorylated, annealed and ligated into the linearized backbone using T4 ligase (NEB, cat. no. M0202S). sgRNA DMXL1 GCTAGCAAAGAACCATCCAT sgRNA DMXL2 GTGACATTCTATAAAGATTG sgRNA WDR7 GGCAGCATTAGACCATGAAG dTAG-3xHA-P2A-Puro sequence: GGAGGCGGTTCAGGGGTACAGGTCGAGACAATAAGTCCGGGGGACGGTAGAACTTTCCCCAAAAGGGGCCAGACGTGTGTTGTGCATTATACGGGCATGTTGGAGGATGGAAAGAAAGTAGACTCCTCACGGGATAGAAACAAACCCTTTAAATTTATGTTGGGGAAGCAAGAGGTTATTAGGGGATGGGAAGAGGGTGTAGCCCAGATGTCTGTAGGCCAACGCGCAAAATTGACTATAAGTCCAGATTATGCGTACGGCGCTACCGGTCACCCTGGGATAATTCCCCCACACGCCACGCTGGTGTTTGATGTCGAACTTCTCAAACTGGAAGGCTCTGGTTATCCGTACGATGTAC- CTGATTATGCCGGGTATCCCTACGACGTGCCAGACTATGCTGGTTATCCGTATGACGTTCCGGACTATGCTGGTGCGACCAATTTTAGTTTGTTGAAACAGGCGGGTGATGTGGAGGAAAACCCGGGGCCTATGACCGAGTATAAACCCACCGTAAGACTGGCGACCAGGGACGACGTTCCAAGGGCTGTCCGGACGCTGGCTGCTGCGTTCGCGGATTACCCTGCCACTAGGCACACGGTTGACCCCGATCGACATATTGAAAGGGTAACTGAACTTCAAGAGTTGTTCCTGACGCGGGTAGGTCTGGATATAGGAAAAGTATGGGTAGCTGATGATGGCGCGGCTGTGGCAGTCTGGACCACTCCGGAAAGCGTTGAAGCTGGCGCAGTTTTCGCTGAAATCGGTCCCAGGATGGCAGAACTTAGTGGGTCCCGATTGGCGGCACAACAGCAAATGGAGGGACTGCTTGCACCGCACCGGCCTAAAGAGCCCGCGTGGTTCCTTGCCACCGTAGGTGTATCTCCTGATCACCAGGGAAAAGGTCTTGGCTCCGCCGTTGTGCTCCCGGGTGTTGAGGCTGCCGAAAGAGCAGGAGTCCCTGCATTTTTGGAAACTAGTGCCCCGCGAAACCTCCCTTTTTACGAACGCTTGGGTTTCACAGTCACGGCCGACGTTGAAGTCCCAGAGGGACCCAGAACGTGGTGCATGACCAGGAAACCCGGGGCT Helical bundle domain amino acid sequence: MYPYDVPDYAGGGSGQEHARVLSSHLMHSSLPGLTRLEQMFLVALADTVATTSTELDESRDKSCSGRDTLDECGLRYLLAMRLHTCLLTSLPPLYRVQLLHQGVSTCHFAWAFHSEAEEELINMIPAIQRGDPQWSELRAMGIGWWVRNINTLRRCIEKVAKASFQRNNDALDAALFYLSMKKKAVVWGLFRSQHDEKMTTFFSHNFNEDRWRKAALKNAFSLLGKQRFEQSAAFFLLAGSLKDAIEVCLEKMEDIQLAMVIARLYESEFETSSTYISILNQKILGCQKDGSGFSCKRLHPDPFLRSLAYWVMKDYTRALDTLLEQTPKEDDEHQVIIKSCNPVAFSFYNYLRTHPLLIRRNLASPEGTLATLGLKTEKNFVDKINLIERKLFFTTANAHFKVGCPVLALEVLSKI HA-DMXL2 WT or mutants were PCR amplified from the pRK5 vector. ..
Amplification:Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients
Article Snippet: .. To construct the lentiviral CRISPR–Cas9 vectors containing the desired sgRNA, the lentiCRISPRv2 backbone (Addgene, 52961) was digested with BsmBI. sgRNA oligos, which included CACC or AAAC overhangs, were synthesized by IDT, phosphorylated, annealed and ligated into the linearized backbone using T4 ligase (NEB, cat. no. M0202S). sgRNA DMXL1 GCTAGCAAAGAACCATCCAT sgRNA DMXL2 GTGACATTCTATAAAGATTG sgRNA WDR7 GGCAGCATTAGACCATGAAG dTAG-3xHA-P2A-Puro sequence: GGAGGCGGTTCAGGGGTACAGGTCGAGACAATAAGTCCGGGGGACGGTAGAACTTTCCCCAAAAGGGGCCAGACGTGTGTTGTGCATTATACGGGCATGTTGGAGGATGGAAAGAAAGTAGACTCCTCACGGGATAGAAACAAACCCTTTAAATTTATGTTGGGGAAGCAAGAGGTTATTAGGGGATGGGAAGAGGGTGTAGCCCAGATGTCTGTAGGCCAACGCGCAAAATTGACTATAAGTCCAGATTATGCGTACGGCGCTACCGGTCACCCTGGGATAATTCCCCCACACGCCACGCTGGTGTTTGATGTCGAACTTCTCAAACTGGAAGGCTCTGGTTATCCGTACGATGTACCTGATTATGCCGGGTATCCCTACGACGTGCCAGACTATGCTGGTTATCCGTATGACGTTCCGGACTATGCTGGTGCGACCAATTTTAGTTTGTTGAAACAGGCGGGTGATGTGGAGGAAAACCCGGGGCCTATGACCGAGTATAAACCCACCGTAAGACTGGCGACCAGGGACGACGTTCCAAGGGCTGTCCGGACGCTGGCTGCTGCGTTCGCGGATTACCCTGCCACTAGGCACACGGTTGACCCCGATCGACATATTGAAAGGGTAACTGAACTTCAAGAGTTGTTCCTGACGCGGGTAGGTCTGGATATAGGAAAAGTATGGGTAGCTGATGATGGCGCGGCTGTGGCAGTCTGGACCACTCCGGAAAGCGTTGAAGCTGGCGCAGTTTTCGCTGAAATCGGTCCCAGGATGGCAGAACTTAGTGGGTCCCGATTGGCGGCACAACAGCAAATGGAGGGACTGCTTGCACCGCACCGGCCTAAAGAGCCCGCGTGGTTCCTTGCCACCGTAGGTGTATCTCCTGATCACCAGGGAAAAGGTCTTGGCTCCGCCGTTGTGCTCCCGGGTGTTGAGGCTGCCGAAAGAGCAGGAGTCCCTGCATTTTTGGAAACTAGTGCCCCGCGAAACCTCCCTTTTTACGAACGCTTGGGTTTCACAGTCACGGCCGACGTTGAAGTCCCAGAGGGACCCAGAACGTGGTGCATGACCAGGAAACCCGGGGCT Helical bundle domain amino acid sequence: MYPYDVPDYAGGGSGQEHARVLSSHLMHSSLPGLTRLEQMFLVALADTVATTSTELDESRDKSCSGRDTLDECGLRYLLAMRLHTCLLTSLPPLYRVQLLHQGVSTCHFAWAFHSEAEEELINMIPAIQRGDPQWSELRAMGIGWWVRNINTLRRCIEKVAKASFQRNNDALDAALFYLSMKKKAVVWGLFRSQHDEKMTTFFSHNFNEDRWRKAALKNAFSLLGKQRFEQSAAFFLLAGSLKDAIEVCLEKMEDIQLAMVIARLYESEFETSSTYISILNQKILGCQKDGSGFSCKRLHPDPFLRSLAYWVMKDYTRALDTLLEQTPKEDDEHQVIIKSCNPVAFSFYNYLRTHPLLIRRNLASPEGTLATLGLKTEKNFVDKINLIERKLFFTTANAHFKVGCPVLALEVLSKI HA-DMXL2 WT or mutants were PCR amplified from the pRK5 vector. ..
Article Title: A heterotrimeric protein complex assembles the metazoan V-ATPase upon dissipation of proton gradients.
Article Snippet: .. To construct the lentiviral CRISPR–Cas9 vectors containing the desired sgRNA, the lentiCRISPRv2 backbone (Addgene, 52961) was digested with BsmBI. sgRNA oligos, which included CACC or AAAC overhangs, were synthesized by IDT, phosphorylated, annealed and ligated into the linearized backbone using T4 ligase (NEB, cat. no. M0202S). sgRNA DMXL1 GCTAGCAAAGAACCATCCAT sgRNA DMXL2 GTGACATTCTATAAAGATTG sgRNA WDR7 GGCAGCATTAGACCATGAAG dTAG-3xHA-P2A-Puro sequence: GGAGGCGGTTCAGGGGTACAGGTCGAGACAATAAGTCCGGGGGACGGTAGAACTTTCCCCAAAAGGGGCCAGACGTGTGTTGTGCATTATACGGGCATGTTGGAGGATGGAAAGAAAGTAGACTCCTCACGGGATAGAAACAAACCCTTTAAATTTATGTTGGGGAAGCAAGAGGTTATTAGGGGATGGGAAGAGGGTGTAGCCCAGATGTCTGTAGGCCAACGCGCAAAATTGACTATAAGTCCAGATTATGCGTACGGCGCTACCGGTCACCCTGGGATAATTCCCCCACACGCCACGCTGGTGTTTGATGTCGAACTTCTCAAACTGGAAGGCTCTGGTTATCCGTACGATGTAC- CTGATTATGCCGGGTATCCCTACGACGTGCCAGACTATGCTGGTTATCCGTATGACGTTCCGGACTATGCTGGTGCGACCAATTTTAGTTTGTTGAAACAGGCGGGTGATGTGGAGGAAAACCCGGGGCCTATGACCGAGTATAAACCCACCGTAAGACTGGCGACCAGGGACGACGTTCCAAGGGCTGTCCGGACGCTGGCTGCTGCGTTCGCGGATTACCCTGCCACTAGGCACACGGTTGACCCCGATCGACATATTGAAAGGGTAACTGAACTTCAAGAGTTGTTCCTGACGCGGGTAGGTCTGGATATAGGAAAAGTATGGGTAGCTGATGATGGCGCGGCTGTGGCAGTCTGGACCACTCCGGAAAGCGTTGAAGCTGGCGCAGTTTTCGCTGAAATCGGTCCCAGGATGGCAGAACTTAGTGGGTCCCGATTGGCGGCACAACAGCAAATGGAGGGACTGCTTGCACCGCACCGGCCTAAAGAGCCCGCGTGGTTCCTTGCCACCGTAGGTGTATCTCCTGATCACCAGGGAAAAGGTCTTGGCTCCGCCGTTGTGCTCCCGGGTGTTGAGGCTGCCGAAAGAGCAGGAGTCCCTGCATTTTTGGAAACTAGTGCCCCGCGAAACCTCCCTTTTTACGAACGCTTGGGTTTCACAGTCACGGCCGACGTTGAAGTCCCAGAGGGACCCAGAACGTGGTGCATGACCAGGAAACCCGGGGCT Helical bundle domain amino acid sequence: MYPYDVPDYAGGGSGQEHARVLSSHLMHSSLPGLTRLEQMFLVALADTVATTSTELDESRDKSCSGRDTLDECGLRYLLAMRLHTCLLTSLPPLYRVQLLHQGVSTCHFAWAFHSEAEEELINMIPAIQRGDPQWSELRAMGIGWWVRNINTLRRCIEKVAKASFQRNNDALDAALFYLSMKKKAVVWGLFRSQHDEKMTTFFSHNFNEDRWRKAALKNAFSLLGKQRFEQSAAFFLLAGSLKDAIEVCLEKMEDIQLAMVIARLYESEFETSSTYISILNQKILGCQKDGSGFSCKRLHPDPFLRSLAYWVMKDYTRALDTLLEQTPKEDDEHQVIIKSCNPVAFSFYNYLRTHPLLIRRNLASPEGTLATLGLKTEKNFVDKINLIERKLFFTTANAHFKVGCPVLALEVLSKI HA-DMXL2 WT or mutants were PCR amplified from the pRK5 vector. ..
Cloning:Article Title: Radiation-induced nuclear translocation of NPRL2 hijacks E3 ubiquitin ligases to enhance DNA repair via the AMPK/WDR24 axis, contributing to CRC radioresistance
Article Snippet: .. The CRISPR-KO cell lines were constructed by cloning single-guide RNAs (sgRNAs) targeting NPRL2 (sg: 5′-GCAGAGGCTGGCCATACCAA-3′), DEPDC5 (sg: 5′-AAGGATCAGTATATTGGCCG-3′), WDR59 (sg: 5′-GGGACATTGGAGCTGTGCAG-3′), SEC13 (sg: 5′-GGTGTCAGTAATTAACACTG-3′), WDR24 (5′-GCACAGCTCAGGTTAAGCGA-3′), PRKAA1 (the gene encoding AMPK α 1, sg: 5′-GCAACTATCGATCTTGCCAA-3′), PRKAA2 (the gene encoding AMPK α 2, sg: 5′-GAAGAGTTGAGTACGCGCTC-3′) and Negative Control (Scrambled, sg: 5′-CGAGCTTGACTACTTGGCAA-3′) into the lentiCRISPRv2 backbone (Addgene [Watertown, MA, USA] plasmid No. 52961). ..
Article Title: Radiation-induced nuclear translocation of NPRL2 hijacks E3 ubiquitin ligases to enhance DNA repair via the AMPK/WDR24 axis, contributing to CRC radioresistance
Article Snippet: .. The CRISPR-KO cell lines were constructed by cloning singleguide RNAs (sgRNAs) targeting NPRL2 (sg: 5 ′ -GCAGAGGCTGGCCATACCAA-3 ′ ), DEPDC5 (sg: 5 ′ -AAGGATCAGTATATTGGCCG-3 ′ ), WDR59 (sg: 5 ′ -GGGACATTGGAGC TGTGCAG-3 ′ ), SEC13 (sg: 5 ′ -GGTGTCAGTAATTAACACTG3 ′ ), WDR24 (5 ′ -GCACAGCTCAGGTTAAGCGA-3 ′ ), PRKAA1 (the gene encoding AMPKα1, sg: 5 ′ -GCAACTATCGATCTTG CCAA-3 ′ ), PRKAA2 (the gene encoding AMPKα2, sg: 5 ′ -GAAGAGTTGAGTACGCGCTC-3 ′ ) and Negative Control (Scrambled, sg: 5 ′ -CGAGCTTGACTACTTGGCAA-3 ′ ) into the lentiCRISPRv2 backbone (Addgene [Watertown, MA, USA] plasmid No. 52961). ..
Negative Control:Article Title: Radiation-induced nuclear translocation of NPRL2 hijacks E3 ubiquitin ligases to enhance DNA repair via the AMPK/WDR24 axis, contributing to CRC radioresistance
Article Snippet: .. The CRISPR-KO cell lines were constructed by cloning single-guide RNAs (sgRNAs) targeting NPRL2 (sg: 5′-GCAGAGGCTGGCCATACCAA-3′), DEPDC5 (sg: 5′-AAGGATCAGTATATTGGCCG-3′), WDR59 (sg: 5′-GGGACATTGGAGCTGTGCAG-3′), SEC13 (sg: 5′-GGTGTCAGTAATTAACACTG-3′), WDR24 (5′-GCACAGCTCAGGTTAAGCGA-3′), PRKAA1 (the gene encoding AMPK α 1, sg: 5′-GCAACTATCGATCTTGCCAA-3′), PRKAA2 (the gene encoding AMPK α 2, sg: 5′-GAAGAGTTGAGTACGCGCTC-3′) and Negative Control (Scrambled, sg: 5′-CGAGCTTGACTACTTGGCAA-3′) into the lentiCRISPRv2 backbone (Addgene [Watertown, MA, USA] plasmid No. 52961). ..
Article Title: Radiation-induced nuclear translocation of NPRL2 hijacks E3 ubiquitin ligases to enhance DNA repair via the AMPK/WDR24 axis, contributing to CRC radioresistance
Article Snippet: .. The CRISPR-KO cell lines were constructed by cloning singleguide RNAs (sgRNAs) targeting NPRL2 (sg: 5 ′ -GCAGAGGCTGGCCATACCAA-3 ′ ), DEPDC5 (sg: 5 ′ -AAGGATCAGTATATTGGCCG-3 ′ ), WDR59 (sg: 5 ′ -GGGACATTGGAGC TGTGCAG-3 ′ ), SEC13 (sg: 5 ′ -GGTGTCAGTAATTAACACTG3 ′ ), WDR24 (5 ′ -GCACAGCTCAGGTTAAGCGA-3 ′ ), PRKAA1 (the gene encoding AMPKα1, sg: 5 ′ -GCAACTATCGATCTTG CCAA-3 ′ ), PRKAA2 (the gene encoding AMPKα2, sg: 5 ′ -GAAGAGTTGAGTACGCGCTC-3 ′ ) and Negative Control (Scrambled, sg: 5 ′ -CGAGCTTGACTACTTGGCAA-3 ′ ) into the lentiCRISPRv2 backbone (Addgene [Watertown, MA, USA] plasmid No. 52961). ..
Plasmid Preparation:Article Title: Radiation-induced nuclear translocation of NPRL2 hijacks E3 ubiquitin ligases to enhance DNA repair via the AMPK/WDR24 axis, contributing to CRC radioresistance
Article Snippet: .. The CRISPR-KO cell lines were constructed by cloning single-guide RNAs (sgRNAs) targeting NPRL2 (sg: 5′-GCAGAGGCTGGCCATACCAA-3′), DEPDC5 (sg: 5′-AAGGATCAGTATATTGGCCG-3′), WDR59 (sg: 5′-GGGACATTGGAGCTGTGCAG-3′), SEC13 (sg: 5′-GGTGTCAGTAATTAACACTG-3′), WDR24 (5′-GCACAGCTCAGGTTAAGCGA-3′), PRKAA1 (the gene encoding AMPK α 1, sg: 5′-GCAACTATCGATCTTGCCAA-3′), PRKAA2 (the gene encoding AMPK α 2, sg: 5′-GAAGAGTTGAGTACGCGCTC-3′) and Negative Control (Scrambled, sg: 5′-CGAGCTTGACTACTTGGCAA-3′) into the lentiCRISPRv2 backbone (Addgene [Watertown, MA, USA] plasmid No. 52961). ..
Article Title: Radiation-induced nuclear translocation of NPRL2 hijacks E3 ubiquitin ligases to enhance DNA repair via the AMPK/WDR24 axis, contributing to CRC radioresistance
Article Snippet: .. The CRISPR-KO cell lines were constructed by cloning singleguide RNAs (sgRNAs) targeting NPRL2 (sg: 5 ′ -GCAGAGGCTGGCCATACCAA-3 ′ ), DEPDC5 (sg: 5 ′ -AAGGATCAGTATATTGGCCG-3 ′ ), WDR59 (sg: 5 ′ -GGGACATTGGAGC TGTGCAG-3 ′ ), SEC13 (sg: 5 ′ -GGTGTCAGTAATTAACACTG3 ′ ), WDR24 (5 ′ -GCACAGCTCAGGTTAAGCGA-3 ′ ), PRKAA1 (the gene encoding AMPKα1, sg: 5 ′ -GCAACTATCGATCTTG CCAA-3 ′ ), PRKAA2 (the gene encoding AMPKα2, sg: 5 ′ -GAAGAGTTGAGTACGCGCTC-3 ′ ) and Negative Control (Scrambled, sg: 5 ′ -CGAGCTTGACTACTTGGCAA-3 ′ ) into the lentiCRISPRv2 backbone (Addgene [Watertown, MA, USA] plasmid No. 52961). ..
Article Title: Cell heterogeneity and fate bistability drive tissue patterning during intestinal regeneration
Article Snippet: After that maintenance of organoid cultures was performed in IntestiCult Organoid Growth Medium (STEMCELL Technologies) with 100μg/ml Penicillin-Streptomycin. .. For the CRISPR-Cas9 mediated knockout of FOXA1, 20bp long guide sequences were designed using the online CHOPCHOP tool ( https://chopchop.cbu.uib.no ) ( ) and cloned into the lentiCRISPRv2 backbone (Addgene plasmid #52961) using the protocol described previously ( ). .. Briefly, vectors were digested using BsmBI and used for ligation with the phosphorylated sgRNA oligos.
Clone Assay:Article Title: Oxidative stress sensing by the translation elongation machinery promotes production of detoxifying selenoproteins
Article Snippet: Flow cytometry data were processed and visualized using FlowJo software (version 10.10.0; BD Biosciences). .. Single guide RNA (sgRNA) sequences targeting genes of interest were cloned into the lentiCRISPRv2 backbone (Addgene #52961) using NEBuilder HiFi DNA Assembly (New England Biolabs). ..
Article Title: Cell heterogeneity and fate bistability drive tissue patterning during intestinal regeneration
Article Snippet: After that maintenance of organoid cultures was performed in IntestiCult Organoid Growth Medium (STEMCELL Technologies) with 100μg/ml Penicillin-Streptomycin. .. For the CRISPR-Cas9 mediated knockout of FOXA1, 20bp long guide sequences were designed using the online CHOPCHOP tool ( https://chopchop.cbu.uib.no ) ( ) and cloned into the lentiCRISPRv2 backbone (Addgene plasmid #52961) using the protocol described previously ( ). .. Briefly, vectors were digested using BsmBI and used for ligation with the phosphorylated sgRNA oligos.
Knock-Out:Article Title: Cell heterogeneity and fate bistability drive tissue patterning during intestinal regeneration
Article Snippet: After that maintenance of organoid cultures was performed in IntestiCult Organoid Growth Medium (STEMCELL Technologies) with 100μg/ml Penicillin-Streptomycin. .. For the CRISPR-Cas9 mediated knockout of FOXA1, 20bp long guide sequences were designed using the online CHOPCHOP tool ( https://chopchop.cbu.uib.no ) ( ) and cloned into the lentiCRISPRv2 backbone (Addgene plasmid #52961) using the protocol described previously ( ). .. Briefly, vectors were digested using BsmBI and used for ligation with the phosphorylated sgRNA oligos.
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