Journal: STAR Protocols

Article Title: Protocol for detecting genomic insulators in Drosophila using insulator-seq, a massively parallel reporter assay

doi: 10.1016/j.xpro.2024.103391

Figure Lengend Snippet: Barcode oligo design The parts of the insulator-seq barcode oligos are shown. The color code used for gene-identifying sequences was kept in all figures.

Article Snippet: Insulator-seq reporter library cloning (A) Barcode oligo pool is amplified by PCR (step 1a). (B) Amplified barcode oligo pool and vector (insulator-seq barcode vector; Addgene 221458) are digested with BsaI (step 1b). (C) BsaI-digested barcode oligo pool and vector are ligated, preserving BsaI-recognition site (step 1c). (D) Result of cloning step 1: single barcode plasmid (step 1d). (E) Single barcode plasmid pool is digested with BsaI (step 2a). (F) BsaI-digested barcode oligo pool and BbsI-digested single barcode plasmid pool are ligated. (G) Result of cloning step 2: Double barcode plasmid pool. (H) 3′UTR-Enhancer-Neutral DNA-3′UTR sequence (Addgene 221459) is inserted between the double barcodes by BbsI-mediated Golden Gate assembly (step 3a). (I) Result of cloning step 3: double barcodes separated by the enhancer, neutral DNA, parts of 3′UTR sequences. (J) Double barcode and enhancer insert pool is inserted between the reporter genes (Addgene 221460) by BsaI-mediated Golden Gate assembly (step 4a). (K) Result of cloning step 3: insulator-seq acceptor library. (L) Insulator-seq insert is generated by tagmenting DNA of the locus of interest (step 5). (M) Insulator-seq insert is generated by PCR-amplifying designer oligo pool library (step 6). (N) Insulator-seq insert is assembled into BamHI-linearized insulator-seq acceptor library by Gibson assembly (step 7). (O) Product of cloning step 7 – insulator-seq reporter library.

Techniques:

Journal: STAR Protocols

Article Title: Protocol for detecting genomic insulators in Drosophila using insulator-seq, a massively parallel reporter assay

doi: 10.1016/j.xpro.2024.103391

Figure Lengend Snippet: Insulator-seq reporter library cloning (A) Barcode oligo pool is amplified by PCR (step 1a). (B) Amplified barcode oligo pool and vector (insulator-seq barcode vector; Addgene 221458) are digested with BsaI (step 1b). (C) BsaI-digested barcode oligo pool and vector are ligated, preserving BsaI-recognition site (step 1c). (D) Result of cloning step 1: single barcode plasmid (step 1d). (E) Single barcode plasmid pool is digested with BsaI (step 2a). (F) BsaI-digested barcode oligo pool and BbsI-digested single barcode plasmid pool are ligated. (G) Result of cloning step 2: Double barcode plasmid pool. (H) 3′UTR-Enhancer-Neutral DNA-3′UTR sequence (Addgene 221459) is inserted between the double barcodes by BbsI-mediated Golden Gate assembly (step 3a). (I) Result of cloning step 3: double barcodes separated by the enhancer, neutral DNA, parts of 3′UTR sequences. (J) Double barcode and enhancer insert pool is inserted between the reporter genes (Addgene 221460) by BsaI-mediated Golden Gate assembly (step 4a). (K) Result of cloning step 3: insulator-seq acceptor library. (L) Insulator-seq insert is generated by tagmenting DNA of the locus of interest (step 5). (M) Insulator-seq insert is generated by PCR-amplifying designer oligo pool library (step 6). (N) Insulator-seq insert is assembled into BamHI-linearized insulator-seq acceptor library by Gibson assembly (step 7). (O) Product of cloning step 7 – insulator-seq reporter library.

Article Snippet: Insulator-seq reporter library cloning (A) Barcode oligo pool is amplified by PCR (step 1a). (B) Amplified barcode oligo pool and vector (insulator-seq barcode vector; Addgene 221458) are digested with BsaI (step 1b). (C) BsaI-digested barcode oligo pool and vector are ligated, preserving BsaI-recognition site (step 1c). (D) Result of cloning step 1: single barcode plasmid (step 1d). (E) Single barcode plasmid pool is digested with BsaI (step 2a). (F) BsaI-digested barcode oligo pool and BbsI-digested single barcode plasmid pool are ligated. (G) Result of cloning step 2: Double barcode plasmid pool. (H) 3′UTR-Enhancer-Neutral DNA-3′UTR sequence (Addgene 221459) is inserted between the double barcodes by BbsI-mediated Golden Gate assembly (step 3a). (I) Result of cloning step 3: double barcodes separated by the enhancer, neutral DNA, parts of 3′UTR sequences. (J) Double barcode and enhancer insert pool is inserted between the reporter genes (Addgene 221460) by BsaI-mediated Golden Gate assembly (step 4a). (K) Result of cloning step 3: insulator-seq acceptor library. (L) Insulator-seq insert is generated by tagmenting DNA of the locus of interest (step 5). (M) Insulator-seq insert is generated by PCR-amplifying designer oligo pool library (step 6). (N) Insulator-seq insert is assembled into BamHI-linearized insulator-seq acceptor library by Gibson assembly (step 7). (O) Product of cloning step 7 – insulator-seq reporter library.

Techniques: Clone Assay, Amplification, Plasmid Preparation, Preserving, Sequencing, Generated

Journal: STAR Protocols

Article Title: Protocol for detecting genomic insulators in Drosophila using insulator-seq, a massively parallel reporter assay

doi: 10.1016/j.xpro.2024.103391

Figure Lengend Snippet:

Article Snippet: Insulator-seq reporter library cloning (A) Barcode oligo pool is amplified by PCR (step 1a). (B) Amplified barcode oligo pool and vector (insulator-seq barcode vector; Addgene 221458) are digested with BsaI (step 1b). (C) BsaI-digested barcode oligo pool and vector are ligated, preserving BsaI-recognition site (step 1c). (D) Result of cloning step 1: single barcode plasmid (step 1d). (E) Single barcode plasmid pool is digested with BsaI (step 2a). (F) BsaI-digested barcode oligo pool and BbsI-digested single barcode plasmid pool are ligated. (G) Result of cloning step 2: Double barcode plasmid pool. (H) 3′UTR-Enhancer-Neutral DNA-3′UTR sequence (Addgene 221459) is inserted between the double barcodes by BbsI-mediated Golden Gate assembly (step 3a). (I) Result of cloning step 3: double barcodes separated by the enhancer, neutral DNA, parts of 3′UTR sequences. (J) Double barcode and enhancer insert pool is inserted between the reporter genes (Addgene 221460) by BsaI-mediated Golden Gate assembly (step 4a). (K) Result of cloning step 3: insulator-seq acceptor library. (L) Insulator-seq insert is generated by tagmenting DNA of the locus of interest (step 5). (M) Insulator-seq insert is generated by PCR-amplifying designer oligo pool library (step 6). (N) Insulator-seq insert is assembled into BamHI-linearized insulator-seq acceptor library by Gibson assembly (step 7). (O) Product of cloning step 7 – insulator-seq reporter library.

Techniques: