Multiplex Assay:Article Title: A novel human model to deconvolve cell-intrinsic phenotypes of genetically dysregulated pathways in lung squamous cell carcinoma
Article Snippet: .. The one-step multiplex CRISPR-Cas9 assembly system kit was a gift from Takashi Yamamoto (Addgene Kit #1000000055) ( ) was used to construct multiplex CRISPR-Cas9 plasmids used for the generation of cells harbouring knockouts in TP53, CDKN2A , PTEN and KEAP1 genes. gRNAs were assembled into gRNA expression cassettes within pX330 plasmids using golden gate cloning, as described previously ( ). ..
Article Title: A human model to deconvolve genotype-phenotype causations in lung squamous cell carcinoma
Article Snippet: .. The one-step multiplex CRISPR-Cas9 assembly system kit was a gift from Takashi Yamamoto (Addgene Kit #1000000055) was used to construct multiplex CRISPR-Cas9 plasmids used for the generation of cells harbouring knockouts in TP53, CDKN2A , PTEN and KEAP1 genes. gRNAs were assembled into gRNA expression cassettes within pX330 plasmids using the golden gate cloning developed by Sakuma et al. . Firstly, each pair of complementary oligonucleotides (Supplementary Table ) were annealed by combining 1 μl each of forward and reverse oligonucleotides (100 μM), 1 μl of T4 DNA Ligase Reaction Buffer (NEB, B0202), and 0.5 μl T4 Polynucleotide Kinase (NEB, M0201) in Ultrapure water to a total volume of 10 μl and annealed using a thermocycler (37 °C for 30 min, 95 °C for 5 min, ramp down to 25 °C at 5 °C/minute). .. Oligonucleotides were then cloned into the relevant pX330S-(2-4)plasmids by combining 0.3 μl of 25 ng/μl plasmid, 0.5 μl of annealed oligonucleotides, 0.2 μl of T4 DNA Ligase Reaction Buffer (NEB, B0202), 0.1 μl Bpil restriction enzyme (Thermo Fisher Scientific, ER1011) and 0.1 μl of Quick Ligase (NEB, M2200) and made to 2 μl with Ultrapure water.
Article Title: A human model to deconvolve genotype-phenotype causations in lung squamous cell carcinoma.
Article Snippet: .. The one-step multiplex CRISPR-Cas9 assembly system kit was a gift from Takashi Yamamoto (Addgene Kit #1000000055)44 was used to construct multiplex CRISPR-Cas9 plasmids used for the generation of cells harbouring knockouts inTP53, CDKN2A, PTEN andKEAP1genes. gRNAs were assembled into gRNA expression cassettes within pX330 plasmids using the golden gate cloning developed by Sakuma et al.44. .. Firstly, each pair of complementary oligonucleotides (Supplementary Table 2) were annealed by combining 1 μl each of forward and reverse oligonucleotides (100 μM), 1 μl of T4 DNA Ligase Reaction Buffer (NEB, B0202), and0.5 μl T4 Polynucleotide Kinase (NEB,M0201) in Ultrapure water to a total volume of 10 μl and annealed using a thermocycler (37 °C for 30min, 95 °C for 5min, ramp down to 25 °C at 5 °C/minute).
CRISPR:Article Title: A novel human model to deconvolve cell-intrinsic phenotypes of genetically dysregulated pathways in lung squamous cell carcinoma
Article Snippet: .. The one-step multiplex CRISPR-Cas9 assembly system kit was a gift from Takashi Yamamoto (Addgene Kit #1000000055) ( ) was used to construct multiplex CRISPR-Cas9 plasmids used for the generation of cells harbouring knockouts in TP53, CDKN2A , PTEN and KEAP1 genes. gRNAs were assembled into gRNA expression cassettes within pX330 plasmids using golden gate cloning, as described previously ( ). ..
Article Title: A human model to deconvolve genotype-phenotype causations in lung squamous cell carcinoma
Article Snippet: .. The one-step multiplex CRISPR-Cas9 assembly system kit was a gift from Takashi Yamamoto (Addgene Kit #1000000055) was used to construct multiplex CRISPR-Cas9 plasmids used for the generation of cells harbouring knockouts in TP53, CDKN2A , PTEN and KEAP1 genes. gRNAs were assembled into gRNA expression cassettes within pX330 plasmids using the golden gate cloning developed by Sakuma et al. . Firstly, each pair of complementary oligonucleotides (Supplementary Table ) were annealed by combining 1 μl each of forward and reverse oligonucleotides (100 μM), 1 μl of T4 DNA Ligase Reaction Buffer (NEB, B0202), and 0.5 μl T4 Polynucleotide Kinase (NEB, M0201) in Ultrapure water to a total volume of 10 μl and annealed using a thermocycler (37 °C for 30 min, 95 °C for 5 min, ramp down to 25 °C at 5 °C/minute). .. Oligonucleotides were then cloned into the relevant pX330S-(2-4)plasmids by combining 0.3 μl of 25 ng/μl plasmid, 0.5 μl of annealed oligonucleotides, 0.2 μl of T4 DNA Ligase Reaction Buffer (NEB, B0202), 0.1 μl Bpil restriction enzyme (Thermo Fisher Scientific, ER1011) and 0.1 μl of Quick Ligase (NEB, M2200) and made to 2 μl with Ultrapure water.
Article Title: A human model to deconvolve genotype-phenotype causations in lung squamous cell carcinoma.
Article Snippet: .. The one-step multiplex CRISPR-Cas9 assembly system kit was a gift from Takashi Yamamoto (Addgene Kit #1000000055)44 was used to construct multiplex CRISPR-Cas9 plasmids used for the generation of cells harbouring knockouts inTP53, CDKN2A, PTEN andKEAP1genes. gRNAs were assembled into gRNA expression cassettes within pX330 plasmids using the golden gate cloning developed by Sakuma et al.44. .. Firstly, each pair of complementary oligonucleotides (Supplementary Table 2) were annealed by combining 1 μl each of forward and reverse oligonucleotides (100 μM), 1 μl of T4 DNA Ligase Reaction Buffer (NEB, B0202), and0.5 μl T4 Polynucleotide Kinase (NEB,M0201) in Ultrapure water to a total volume of 10 μl and annealed using a thermocycler (37 °C for 30min, 95 °C for 5min, ramp down to 25 °C at 5 °C/minute).
Construct:Article Title: A novel human model to deconvolve cell-intrinsic phenotypes of genetically dysregulated pathways in lung squamous cell carcinoma
Article Snippet: .. The one-step multiplex CRISPR-Cas9 assembly system kit was a gift from Takashi Yamamoto (Addgene Kit #1000000055) ( ) was used to construct multiplex CRISPR-Cas9 plasmids used for the generation of cells harbouring knockouts in TP53, CDKN2A , PTEN and KEAP1 genes. gRNAs were assembled into gRNA expression cassettes within pX330 plasmids using golden gate cloning, as described previously ( ). ..
Article Title: A human model to deconvolve genotype-phenotype causations in lung squamous cell carcinoma
Article Snippet: .. The one-step multiplex CRISPR-Cas9 assembly system kit was a gift from Takashi Yamamoto (Addgene Kit #1000000055) was used to construct multiplex CRISPR-Cas9 plasmids used for the generation of cells harbouring knockouts in TP53, CDKN2A , PTEN and KEAP1 genes. gRNAs were assembled into gRNA expression cassettes within pX330 plasmids using the golden gate cloning developed by Sakuma et al. . Firstly, each pair of complementary oligonucleotides (Supplementary Table ) were annealed by combining 1 μl each of forward and reverse oligonucleotides (100 μM), 1 μl of T4 DNA Ligase Reaction Buffer (NEB, B0202), and 0.5 μl T4 Polynucleotide Kinase (NEB, M0201) in Ultrapure water to a total volume of 10 μl and annealed using a thermocycler (37 °C for 30 min, 95 °C for 5 min, ramp down to 25 °C at 5 °C/minute). .. Oligonucleotides were then cloned into the relevant pX330S-(2-4)plasmids by combining 0.3 μl of 25 ng/μl plasmid, 0.5 μl of annealed oligonucleotides, 0.2 μl of T4 DNA Ligase Reaction Buffer (NEB, B0202), 0.1 μl Bpil restriction enzyme (Thermo Fisher Scientific, ER1011) and 0.1 μl of Quick Ligase (NEB, M2200) and made to 2 μl with Ultrapure water.
Article Title: A human model to deconvolve genotype-phenotype causations in lung squamous cell carcinoma.
Article Snippet: .. The one-step multiplex CRISPR-Cas9 assembly system kit was a gift from Takashi Yamamoto (Addgene Kit #1000000055)44 was used to construct multiplex CRISPR-Cas9 plasmids used for the generation of cells harbouring knockouts inTP53, CDKN2A, PTEN andKEAP1genes. gRNAs were assembled into gRNA expression cassettes within pX330 plasmids using the golden gate cloning developed by Sakuma et al.44. .. Firstly, each pair of complementary oligonucleotides (Supplementary Table 2) were annealed by combining 1 μl each of forward and reverse oligonucleotides (100 μM), 1 μl of T4 DNA Ligase Reaction Buffer (NEB, B0202), and0.5 μl T4 Polynucleotide Kinase (NEB,M0201) in Ultrapure water to a total volume of 10 μl and annealed using a thermocycler (37 °C for 30min, 95 °C for 5min, ramp down to 25 °C at 5 °C/minute).
Expressing:Article Title: A novel human model to deconvolve cell-intrinsic phenotypes of genetically dysregulated pathways in lung squamous cell carcinoma
Article Snippet: .. The one-step multiplex CRISPR-Cas9 assembly system kit was a gift from Takashi Yamamoto (Addgene Kit #1000000055) ( ) was used to construct multiplex CRISPR-Cas9 plasmids used for the generation of cells harbouring knockouts in TP53, CDKN2A , PTEN and KEAP1 genes. gRNAs were assembled into gRNA expression cassettes within pX330 plasmids using golden gate cloning, as described previously ( ). ..
Article Title: A human model to deconvolve genotype-phenotype causations in lung squamous cell carcinoma
Article Snippet: .. The one-step multiplex CRISPR-Cas9 assembly system kit was a gift from Takashi Yamamoto (Addgene Kit #1000000055) was used to construct multiplex CRISPR-Cas9 plasmids used for the generation of cells harbouring knockouts in TP53, CDKN2A , PTEN and KEAP1 genes. gRNAs were assembled into gRNA expression cassettes within pX330 plasmids using the golden gate cloning developed by Sakuma et al. . Firstly, each pair of complementary oligonucleotides (Supplementary Table ) were annealed by combining 1 μl each of forward and reverse oligonucleotides (100 μM), 1 μl of T4 DNA Ligase Reaction Buffer (NEB, B0202), and 0.5 μl T4 Polynucleotide Kinase (NEB, M0201) in Ultrapure water to a total volume of 10 μl and annealed using a thermocycler (37 °C for 30 min, 95 °C for 5 min, ramp down to 25 °C at 5 °C/minute). .. Oligonucleotides were then cloned into the relevant pX330S-(2-4)plasmids by combining 0.3 μl of 25 ng/μl plasmid, 0.5 μl of annealed oligonucleotides, 0.2 μl of T4 DNA Ligase Reaction Buffer (NEB, B0202), 0.1 μl Bpil restriction enzyme (Thermo Fisher Scientific, ER1011) and 0.1 μl of Quick Ligase (NEB, M2200) and made to 2 μl with Ultrapure water.
Article Title: A human model to deconvolve genotype-phenotype causations in lung squamous cell carcinoma.
Article Snippet: .. The one-step multiplex CRISPR-Cas9 assembly system kit was a gift from Takashi Yamamoto (Addgene Kit #1000000055)44 was used to construct multiplex CRISPR-Cas9 plasmids used for the generation of cells harbouring knockouts inTP53, CDKN2A, PTEN andKEAP1genes. gRNAs were assembled into gRNA expression cassettes within pX330 plasmids using the golden gate cloning developed by Sakuma et al.44. .. Firstly, each pair of complementary oligonucleotides (Supplementary Table 2) were annealed by combining 1 μl each of forward and reverse oligonucleotides (100 μM), 1 μl of T4 DNA Ligase Reaction Buffer (NEB, B0202), and0.5 μl T4 Polynucleotide Kinase (NEB,M0201) in Ultrapure water to a total volume of 10 μl and annealed using a thermocycler (37 °C for 30min, 95 °C for 5min, ramp down to 25 °C at 5 °C/minute).
Cloning:Article Title: A novel human model to deconvolve cell-intrinsic phenotypes of genetically dysregulated pathways in lung squamous cell carcinoma
Article Snippet: .. The one-step multiplex CRISPR-Cas9 assembly system kit was a gift from Takashi Yamamoto (Addgene Kit #1000000055) ( ) was used to construct multiplex CRISPR-Cas9 plasmids used for the generation of cells harbouring knockouts in TP53, CDKN2A , PTEN and KEAP1 genes. gRNAs were assembled into gRNA expression cassettes within pX330 plasmids using golden gate cloning, as described previously ( ). ..
Article Title: A human model to deconvolve genotype-phenotype causations in lung squamous cell carcinoma
Article Snippet: .. The one-step multiplex CRISPR-Cas9 assembly system kit was a gift from Takashi Yamamoto (Addgene Kit #1000000055) was used to construct multiplex CRISPR-Cas9 plasmids used for the generation of cells harbouring knockouts in TP53, CDKN2A , PTEN and KEAP1 genes. gRNAs were assembled into gRNA expression cassettes within pX330 plasmids using the golden gate cloning developed by Sakuma et al. . Firstly, each pair of complementary oligonucleotides (Supplementary Table ) were annealed by combining 1 μl each of forward and reverse oligonucleotides (100 μM), 1 μl of T4 DNA Ligase Reaction Buffer (NEB, B0202), and 0.5 μl T4 Polynucleotide Kinase (NEB, M0201) in Ultrapure water to a total volume of 10 μl and annealed using a thermocycler (37 °C for 30 min, 95 °C for 5 min, ramp down to 25 °C at 5 °C/minute). .. Oligonucleotides were then cloned into the relevant pX330S-(2-4)plasmids by combining 0.3 μl of 25 ng/μl plasmid, 0.5 μl of annealed oligonucleotides, 0.2 μl of T4 DNA Ligase Reaction Buffer (NEB, B0202), 0.1 μl Bpil restriction enzyme (Thermo Fisher Scientific, ER1011) and 0.1 μl of Quick Ligase (NEB, M2200) and made to 2 μl with Ultrapure water.
Article Title: A human model to deconvolve genotype-phenotype causations in lung squamous cell carcinoma.
Article Snippet: .. The one-step multiplex CRISPR-Cas9 assembly system kit was a gift from Takashi Yamamoto (Addgene Kit #1000000055)44 was used to construct multiplex CRISPR-Cas9 plasmids used for the generation of cells harbouring knockouts inTP53, CDKN2A, PTEN andKEAP1genes. gRNAs were assembled into gRNA expression cassettes within pX330 plasmids using the golden gate cloning developed by Sakuma et al.44. .. Firstly, each pair of complementary oligonucleotides (Supplementary Table 2) were annealed by combining 1 μl each of forward and reverse oligonucleotides (100 μM), 1 μl of T4 DNA Ligase Reaction Buffer (NEB, B0202), and0.5 μl T4 Polynucleotide Kinase (NEB,M0201) in Ultrapure water to a total volume of 10 μl and annealed using a thermocycler (37 °C for 30min, 95 °C for 5min, ramp down to 25 °C at 5 °C/minute).
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