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sgrna expression vector pcrispria v2  (Addgene inc)


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    Addgene inc sgrna expression vector pcrispria v2
    Sgrna Expression Vector Pcrispria V2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/sgrna+expression+vector+pcrispria+v2/pCRISPRia-v2+(Plasmid+%2384832)/pm41422267-207-34-38
    Average 96 stars, based on 143 article reviews
    sgrna expression vector pcrispria v2 - by Bioz Stars, 2026-10
    96/100 stars

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    Polymerase Chain Reaction:

    Article Title: Mapping cis - and trans -regulatory target genes of human-specific deletions
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR adapter sequences and restriction sites ( BstXI , BlpI ), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene, 84832; hDel-v1, hDel-v2) or pJR101 (Addgene, 187241; hDel-v3, hDel-v4) as described previously . .. Briefly, oligonucleotide pools were amplified by 8 to 10 cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544X), digested with BstXI and BlpI , size-selected by polyacrylamide gel electrophoresis, ligated into BstXI - and BlpI- digested pCRISPRia-v2 or pJR101, and introduced into MegaX DH10B T1 R cells by electroporation (Invitrogen, C640003; Bio-Rad, 1652660).

    Article Title: Mapping cis- and trans-regulatory target genes of human-specific deletions.
    Article Snippet: .. The complete hDel-v4 sgRNA library contains 923 sgRNAs. hDel sgRNA library cloning Oligonucleotide pools were designed with flanking PCR adapter sequences and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene, 84832; hDel-v1, hDel-v2) or pJR101 (Addgene, 187241; hDel-v3, hDel-v4) as described previously54. .. Briefly, oligonucleotide pools were amplified by 8 to 10 cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544X), digested with BstXI and BlpI, size-selected by polyacrylamide gel electrophoresis, ligated into BstXI- and BlpI-digested pCRISPRia-v2 or pJR101, and introduced intoMegaXDH10BT1R cells by electroporation (Invitrogen, C640003; Bio-Rad, 1652660). hDel CRISPRi screening Chimpanzee CRISPRi iPS cells (C3624K or Pt5-C, hDel-v1; C3624K, hDel-v2) were dissociated with Accutase (Innovative Cell Technologies, AT104-500), resuspended in v3.1 medium supplemented with 2μMthiazovivin and 5μg/mlpolybrene (Mirus,MIR 6620), transduced with the hDel-v1 or hDel-v2 lentiviral sgRNA library at a target infection rate of 25%, and plated at a density of 85,000 cells/cm2 in Matrigelcoated 5-layer cell culture flasks (Corning, 353144).

    Article Title: Comparative landscape of genetic dependencies in human and chimpanzee stem cells
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832) as described previously ( ). ..

    Article Title: Comparative landscape of genetic dependencies in human and chimpanzee stem cells
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832) as described previously ( ). ..

    Article Title: Comparative landscape of genetic dependencies in human and chimpanzee stem cells.
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832) as described previously.32 Pooled validation CRISPRi screening Validation screens were performed in conditions consistent with the genome-wide screens. .. Briefly, CRISPRi PSCs expressing dCas9-KRABwere dissociated with Accutase, resuspended in StemFlex supplemented with 2 mM thiazovivin and 5 mg/ml polybrene, transduced with the lentiviral CEV-v1 sgRNA library at a target infection rate of 25-40%, and plated in Matrigel-coated 3-layer cell culture flasks (Corning, cat. 353143) at a density of 65,000-80,000 cells/cm2.

    Article Title: Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites, synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832), essentially as described previously ( ). ..

    Article Title: Mapping cis - and trans -regulatory target genes of human-specific deletions
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR adapter sequences and restriction sites ( BstXI , BlpI ), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene, 84832; hDel-v1, hDel-v2) or pJR101 (Addgene, 187241; hDel-v3, hDel-v4) as described previously . .. Briefly, oligonucleotide pools were amplified by 8 to 10 cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544X), digested with BstXI and BlpI , size-selected by polyacrylamide gel electrophoresis, ligated into BstXI - and BlpI- digested pCRISPRia-v2 or pJR101, and introduced into MegaX DH10B T1 R cells by electroporation (Invitrogen, C640003; Bio-Rad, 1652660).


    Synthesized:

    Article Title: Mapping cis - and trans -regulatory target genes of human-specific deletions
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR adapter sequences and restriction sites ( BstXI , BlpI ), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene, 84832; hDel-v1, hDel-v2) or pJR101 (Addgene, 187241; hDel-v3, hDel-v4) as described previously . .. Briefly, oligonucleotide pools were amplified by 8 to 10 cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544X), digested with BstXI and BlpI , size-selected by polyacrylamide gel electrophoresis, ligated into BstXI - and BlpI- digested pCRISPRia-v2 or pJR101, and introduced into MegaX DH10B T1 R cells by electroporation (Invitrogen, C640003; Bio-Rad, 1652660).

    Article Title: Mapping cis- and trans-regulatory target genes of human-specific deletions.
    Article Snippet: .. The complete hDel-v4 sgRNA library contains 923 sgRNAs. hDel sgRNA library cloning Oligonucleotide pools were designed with flanking PCR adapter sequences and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene, 84832; hDel-v1, hDel-v2) or pJR101 (Addgene, 187241; hDel-v3, hDel-v4) as described previously54. .. Briefly, oligonucleotide pools were amplified by 8 to 10 cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544X), digested with BstXI and BlpI, size-selected by polyacrylamide gel electrophoresis, ligated into BstXI- and BlpI-digested pCRISPRia-v2 or pJR101, and introduced intoMegaXDH10BT1R cells by electroporation (Invitrogen, C640003; Bio-Rad, 1652660). hDel CRISPRi screening Chimpanzee CRISPRi iPS cells (C3624K or Pt5-C, hDel-v1; C3624K, hDel-v2) were dissociated with Accutase (Innovative Cell Technologies, AT104-500), resuspended in v3.1 medium supplemented with 2μMthiazovivin and 5μg/mlpolybrene (Mirus,MIR 6620), transduced with the hDel-v1 or hDel-v2 lentiviral sgRNA library at a target infection rate of 25%, and plated at a density of 85,000 cells/cm2 in Matrigelcoated 5-layer cell culture flasks (Corning, 353144).

    Article Title: Comparative landscape of genetic dependencies in human and chimpanzee stem cells
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832) as described previously ( ). ..

    Article Title: Comparative landscape of genetic dependencies in human and chimpanzee stem cells
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832) as described previously ( ). ..

    Article Title: Comparative landscape of genetic dependencies in human and chimpanzee stem cells.
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832) as described previously.32 Pooled validation CRISPRi screening Validation screens were performed in conditions consistent with the genome-wide screens. .. Briefly, CRISPRi PSCs expressing dCas9-KRABwere dissociated with Accutase, resuspended in StemFlex supplemented with 2 mM thiazovivin and 5 mg/ml polybrene, transduced with the lentiviral CEV-v1 sgRNA library at a target infection rate of 25-40%, and plated in Matrigel-coated 3-layer cell culture flasks (Corning, cat. 353143) at a density of 65,000-80,000 cells/cm2.

    Article Title: Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites, synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832), essentially as described previously ( ). ..

    Article Title: Mapping cis - and trans -regulatory target genes of human-specific deletions
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR adapter sequences and restriction sites ( BstXI , BlpI ), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene, 84832; hDel-v1, hDel-v2) or pJR101 (Addgene, 187241; hDel-v3, hDel-v4) as described previously . .. Briefly, oligonucleotide pools were amplified by 8 to 10 cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544X), digested with BstXI and BlpI , size-selected by polyacrylamide gel electrophoresis, ligated into BstXI - and BlpI- digested pCRISPRia-v2 or pJR101, and introduced into MegaX DH10B T1 R cells by electroporation (Invitrogen, C640003; Bio-Rad, 1652660).


    Clone Assay:

    Article Title: Mapping cis - and trans -regulatory target genes of human-specific deletions
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR adapter sequences and restriction sites ( BstXI , BlpI ), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene, 84832; hDel-v1, hDel-v2) or pJR101 (Addgene, 187241; hDel-v3, hDel-v4) as described previously . .. Briefly, oligonucleotide pools were amplified by 8 to 10 cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544X), digested with BstXI and BlpI , size-selected by polyacrylamide gel electrophoresis, ligated into BstXI - and BlpI- digested pCRISPRia-v2 or pJR101, and introduced into MegaX DH10B T1 R cells by electroporation (Invitrogen, C640003; Bio-Rad, 1652660).

    Article Title: Mapping cis- and trans-regulatory target genes of human-specific deletions.
    Article Snippet: .. The complete hDel-v4 sgRNA library contains 923 sgRNAs. hDel sgRNA library cloning Oligonucleotide pools were designed with flanking PCR adapter sequences and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene, 84832; hDel-v1, hDel-v2) or pJR101 (Addgene, 187241; hDel-v3, hDel-v4) as described previously54. .. Briefly, oligonucleotide pools were amplified by 8 to 10 cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544X), digested with BstXI and BlpI, size-selected by polyacrylamide gel electrophoresis, ligated into BstXI- and BlpI-digested pCRISPRia-v2 or pJR101, and introduced intoMegaXDH10BT1R cells by electroporation (Invitrogen, C640003; Bio-Rad, 1652660). hDel CRISPRi screening Chimpanzee CRISPRi iPS cells (C3624K or Pt5-C, hDel-v1; C3624K, hDel-v2) were dissociated with Accutase (Innovative Cell Technologies, AT104-500), resuspended in v3.1 medium supplemented with 2μMthiazovivin and 5μg/mlpolybrene (Mirus,MIR 6620), transduced with the hDel-v1 or hDel-v2 lentiviral sgRNA library at a target infection rate of 25%, and plated at a density of 85,000 cells/cm2 in Matrigelcoated 5-layer cell culture flasks (Corning, 353144).

    Article Title: Comparative landscape of genetic dependencies in human and chimpanzee stem cells
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832) as described previously ( ). ..

    Article Title: Comparative landscape of genetic dependencies in human and chimpanzee stem cells
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832) as described previously ( ). ..

    Article Title: Comparative landscape of genetic dependencies in human and chimpanzee stem cells.
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832) as described previously.32 Pooled validation CRISPRi screening Validation screens were performed in conditions consistent with the genome-wide screens. .. Briefly, CRISPRi PSCs expressing dCas9-KRABwere dissociated with Accutase, resuspended in StemFlex supplemented with 2 mM thiazovivin and 5 mg/ml polybrene, transduced with the lentiviral CEV-v1 sgRNA library at a target infection rate of 25-40%, and plated in Matrigel-coated 3-layer cell culture flasks (Corning, cat. 353143) at a density of 65,000-80,000 cells/cm2.

    Article Title: Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites, synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832), essentially as described previously ( ). ..

    Article Title: Mapping cis - and trans -regulatory target genes of human-specific deletions
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR adapter sequences and restriction sites ( BstXI , BlpI ), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene, 84832; hDel-v1, hDel-v2) or pJR101 (Addgene, 187241; hDel-v3, hDel-v4) as described previously . .. Briefly, oligonucleotide pools were amplified by 8 to 10 cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544X), digested with BstXI and BlpI , size-selected by polyacrylamide gel electrophoresis, ligated into BstXI - and BlpI- digested pCRISPRia-v2 or pJR101, and introduced into MegaX DH10B T1 R cells by electroporation (Invitrogen, C640003; Bio-Rad, 1652660).


    Expressing:

    Article Title: Mapping cis - and trans -regulatory target genes of human-specific deletions
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR adapter sequences and restriction sites ( BstXI , BlpI ), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene, 84832; hDel-v1, hDel-v2) or pJR101 (Addgene, 187241; hDel-v3, hDel-v4) as described previously . .. Briefly, oligonucleotide pools were amplified by 8 to 10 cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544X), digested with BstXI and BlpI , size-selected by polyacrylamide gel electrophoresis, ligated into BstXI - and BlpI- digested pCRISPRia-v2 or pJR101, and introduced into MegaX DH10B T1 R cells by electroporation (Invitrogen, C640003; Bio-Rad, 1652660).

    Article Title: Mapping cis- and trans-regulatory target genes of human-specific deletions.
    Article Snippet: .. The complete hDel-v4 sgRNA library contains 923 sgRNAs. hDel sgRNA library cloning Oligonucleotide pools were designed with flanking PCR adapter sequences and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene, 84832; hDel-v1, hDel-v2) or pJR101 (Addgene, 187241; hDel-v3, hDel-v4) as described previously54. .. Briefly, oligonucleotide pools were amplified by 8 to 10 cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544X), digested with BstXI and BlpI, size-selected by polyacrylamide gel electrophoresis, ligated into BstXI- and BlpI-digested pCRISPRia-v2 or pJR101, and introduced intoMegaXDH10BT1R cells by electroporation (Invitrogen, C640003; Bio-Rad, 1652660). hDel CRISPRi screening Chimpanzee CRISPRi iPS cells (C3624K or Pt5-C, hDel-v1; C3624K, hDel-v2) were dissociated with Accutase (Innovative Cell Technologies, AT104-500), resuspended in v3.1 medium supplemented with 2μMthiazovivin and 5μg/mlpolybrene (Mirus,MIR 6620), transduced with the hDel-v1 or hDel-v2 lentiviral sgRNA library at a target infection rate of 25%, and plated at a density of 85,000 cells/cm2 in Matrigelcoated 5-layer cell culture flasks (Corning, 353144).

    Article Title: Comparative landscape of genetic dependencies in human and chimpanzee stem cells
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832) as described previously ( ). ..

    Article Title: Comparative landscape of genetic dependencies in human and chimpanzee stem cells
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832) as described previously ( ). ..

    Article Title: Comparative landscape of genetic dependencies in human and chimpanzee stem cells.
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832) as described previously.32 Pooled validation CRISPRi screening Validation screens were performed in conditions consistent with the genome-wide screens. .. Briefly, CRISPRi PSCs expressing dCas9-KRABwere dissociated with Accutase, resuspended in StemFlex supplemented with 2 mM thiazovivin and 5 mg/ml polybrene, transduced with the lentiviral CEV-v1 sgRNA library at a target infection rate of 25-40%, and plated in Matrigel-coated 3-layer cell culture flasks (Corning, cat. 353143) at a density of 65,000-80,000 cells/cm2.

    Article Title: Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites, synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832), essentially as described previously ( ). ..

    Article Title: Mapping cis - and trans -regulatory target genes of human-specific deletions
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR adapter sequences and restriction sites ( BstXI , BlpI ), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene, 84832; hDel-v1, hDel-v2) or pJR101 (Addgene, 187241; hDel-v3, hDel-v4) as described previously . .. Briefly, oligonucleotide pools were amplified by 8 to 10 cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544X), digested with BstXI and BlpI , size-selected by polyacrylamide gel electrophoresis, ligated into BstXI - and BlpI- digested pCRISPRia-v2 or pJR101, and introduced into MegaX DH10B T1 R cells by electroporation (Invitrogen, C640003; Bio-Rad, 1652660).


    Plasmid Preparation:

    Article Title: Mapping cis - and trans -regulatory target genes of human-specific deletions
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR adapter sequences and restriction sites ( BstXI , BlpI ), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene, 84832; hDel-v1, hDel-v2) or pJR101 (Addgene, 187241; hDel-v3, hDel-v4) as described previously . .. Briefly, oligonucleotide pools were amplified by 8 to 10 cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544X), digested with BstXI and BlpI , size-selected by polyacrylamide gel electrophoresis, ligated into BstXI - and BlpI- digested pCRISPRia-v2 or pJR101, and introduced into MegaX DH10B T1 R cells by electroporation (Invitrogen, C640003; Bio-Rad, 1652660).

    Article Title: Mapping cis- and trans-regulatory target genes of human-specific deletions.
    Article Snippet: .. The complete hDel-v4 sgRNA library contains 923 sgRNAs. hDel sgRNA library cloning Oligonucleotide pools were designed with flanking PCR adapter sequences and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene, 84832; hDel-v1, hDel-v2) or pJR101 (Addgene, 187241; hDel-v3, hDel-v4) as described previously54. .. Briefly, oligonucleotide pools were amplified by 8 to 10 cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544X), digested with BstXI and BlpI, size-selected by polyacrylamide gel electrophoresis, ligated into BstXI- and BlpI-digested pCRISPRia-v2 or pJR101, and introduced intoMegaXDH10BT1R cells by electroporation (Invitrogen, C640003; Bio-Rad, 1652660). hDel CRISPRi screening Chimpanzee CRISPRi iPS cells (C3624K or Pt5-C, hDel-v1; C3624K, hDel-v2) were dissociated with Accutase (Innovative Cell Technologies, AT104-500), resuspended in v3.1 medium supplemented with 2μMthiazovivin and 5μg/mlpolybrene (Mirus,MIR 6620), transduced with the hDel-v1 or hDel-v2 lentiviral sgRNA library at a target infection rate of 25%, and plated at a density of 85,000 cells/cm2 in Matrigelcoated 5-layer cell culture flasks (Corning, 353144).

    Article Title: Mapping cis - and trans -regulatory target genes of human-specific deletions
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR adapter sequences and restriction sites ( BstXI , BlpI ), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene, 84832; hDel-v1, hDel-v2) or pJR101 (Addgene, 187241; hDel-v3, hDel-v4) as described previously . .. Briefly, oligonucleotide pools were amplified by 8 to 10 cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544X), digested with BstXI and BlpI , size-selected by polyacrylamide gel electrophoresis, ligated into BstXI - and BlpI- digested pCRISPRia-v2 or pJR101, and introduced into MegaX DH10B T1 R cells by electroporation (Invitrogen, C640003; Bio-Rad, 1652660).


    Cloning:

    Article Title: Mapping cis- and trans-regulatory target genes of human-specific deletions.
    Article Snippet: .. The complete hDel-v4 sgRNA library contains 923 sgRNAs. hDel sgRNA library cloning Oligonucleotide pools were designed with flanking PCR adapter sequences and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene, 84832; hDel-v1, hDel-v2) or pJR101 (Addgene, 187241; hDel-v3, hDel-v4) as described previously54. .. Briefly, oligonucleotide pools were amplified by 8 to 10 cycles of PCR using NEBNext Ultra II Q5 Master Mix (New England Biolabs, M0544X), digested with BstXI and BlpI, size-selected by polyacrylamide gel electrophoresis, ligated into BstXI- and BlpI-digested pCRISPRia-v2 or pJR101, and introduced intoMegaXDH10BT1R cells by electroporation (Invitrogen, C640003; Bio-Rad, 1652660). hDel CRISPRi screening Chimpanzee CRISPRi iPS cells (C3624K or Pt5-C, hDel-v1; C3624K, hDel-v2) were dissociated with Accutase (Innovative Cell Technologies, AT104-500), resuspended in v3.1 medium supplemented with 2μMthiazovivin and 5μg/mlpolybrene (Mirus,MIR 6620), transduced with the hDel-v1 or hDel-v2 lentiviral sgRNA library at a target infection rate of 25%, and plated at a density of 85,000 cells/cm2 in Matrigelcoated 5-layer cell culture flasks (Corning, 353144).

    Biomarker Discovery:

    Article Title: Comparative landscape of genetic dependencies in human and chimpanzee stem cells.
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832) as described previously.32 Pooled validation CRISPRi screening Validation screens were performed in conditions consistent with the genome-wide screens. .. Briefly, CRISPRi PSCs expressing dCas9-KRABwere dissociated with Accutase, resuspended in StemFlex supplemented with 2 mM thiazovivin and 5 mg/ml polybrene, transduced with the lentiviral CEV-v1 sgRNA library at a target infection rate of 25-40%, and plated in Matrigel-coated 3-layer cell culture flasks (Corning, cat. 353143) at a density of 65,000-80,000 cells/cm2.

    Genome Wide:

    Article Title: Comparative landscape of genetic dependencies in human and chimpanzee stem cells.
    Article Snippet: .. Oligonucleotide pools were designed with flanking PCR and restriction sites (BstXI, BlpI), synthesized by Agilent Technologies, and cloned into the sgRNA expression vector pCRISPRia-v2 (Addgene #84832) as described previously.32 Pooled validation CRISPRi screening Validation screens were performed in conditions consistent with the genome-wide screens. .. Briefly, CRISPRi PSCs expressing dCas9-KRABwere dissociated with Accutase, resuspended in StemFlex supplemented with 2 mM thiazovivin and 5 mg/ml polybrene, transduced with the lentiviral CEV-v1 sgRNA library at a target infection rate of 25-40%, and plated in Matrigel-coated 3-layer cell culture flasks (Corning, cat. 353143) at a density of 65,000-80,000 cells/cm2.



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