Journal: PLOS Pathogens

Article Title: The PRMT5/WDR77 complex restricts hepatitis E virus replication

doi: 10.1371/journal.ppat.1011434

Figure Lengend Snippet: (A) Western blot analysis of lysates from HepG2C3A cells transduced with PRMT5, WDR77 or non-targeting control plentiCRISPRv2 sgRNA lentivirus. Tubulin was used as the loading control. (B) HepG2C3A cells were infected with HEV Kernow C1/p6 virus. Immunofluorescence of ORF2 was performed at day 3 post infection with ORF2 monoclonal antibody (2G8) . (C) Flow cytometry analysis of HEV infection by ORF2 staining using 2G8 antibody was performed at day 3 post infection. (D) RT-qPCR analysis of HEV genomic RNAs at day 3 post infection. (E) Intracellular viral titer of Kernow C1/p6 was determined at day 7 post transfection in S10-3 cells. Values are means plus standard deviations (SD) (error bars) (n = 3). *, P < 0.05; ***, P < 0.001 by one-way ANOVA. All data are representative of three independent experiments.

Article Snippet: plentiCRISPRv2 vector (78852, Addgene) was digested by BsaI restriction enzyme (R3733, NEB) to prepare backbone, then annealed sgRNA oligo was ligated with backbone by T4 DNA ligase (M0202, NEB) to construct knockout sgRNA. sgRNA target sequences are listed in .

Techniques: Western Blot, Transduction, Control, Infection, Virus, Immunofluorescence, Flow Cytometry, Staining, Quantitative RT-PCR, Transfection

Journal: PLOS Pathogens

Article Title: The PRMT5/WDR77 complex restricts hepatitis E virus replication

doi: 10.1371/journal.ppat.1011434

Figure Lengend Snippet: (A) Western blot analysis of lysates from S10-3 cells transduced with PRMT5, WDR77 or non-targeting control plentiCRISPRv2 sgRNA lentivirus. Actin was used as the loading control. (B-D) S10-3 cells were transfected with HEV replicon RNAs of Kernow C1/p6 Gluc replicon (B), Sar55 Gluc replicon (C) and pSHEV3 Gluc replicon (D). Supernatants were collected and Gluc activity was measured at day 2 post transfection. Data are normalized with non-targeting control sgRNA. Values are means plus standard deviations (SD) (error bars) (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001 by one-way ANOVA. All data are representative of three independent experiments.

Article Snippet: plentiCRISPRv2 vector (78852, Addgene) was digested by BsaI restriction enzyme (R3733, NEB) to prepare backbone, then annealed sgRNA oligo was ligated with backbone by T4 DNA ligase (M0202, NEB) to construct knockout sgRNA. sgRNA target sequences are listed in .

Techniques: Western Blot, Transduction, Control, Transfection, Activity Assay

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: