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human pooled genome wide sgrna library brunello sequences addgene addgene  (Addgene inc)


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    Addgene inc human pooled genome wide sgrna library brunello sequences addgene addgene
    Human Pooled Genome Wide Sgrna Library Brunello Sequences Addgene Addgene, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    (A) Workflow of a genome-wide CRISPR screen to identify candidate factors involved in 5′ ss-mediated gene repression. 293 Flp-In cells expressing the eGFP-5′ ss-PAS reporter are infected with a <t>lentiviral</t> <t>Brunello</t> genome-wide sgRNA library and selected with puromycin for 5 days. The selected cells are then treated with doxycycline for 3 days to induce eGFP expression. Following induction, the top 1% of high eGFP-expressing cells are sorted via FACS analysis. Genomic DNA is then extracted, followed by sgRNA barcode amplification and deep sequencing analysis. (B) Candidate genes identified by the CRISPR-Cas9 screen. Data analysis is performed using the MAGeCK algorithm to identify enriched sgRNA, and the genes are ranked by false discovery rate (FDR). Names of top candidates were labeled. (C) A schematic diagram for the RNA pull-down and mass spectrometry analysis. MBP-MS2, a fusion protein combining maltose-binding protein (MBP) and MS2, facilitating RNA capture and purification. 5′ ss: 5′ splicing site; WT: wild type; Mut: mutant. (D) A Venn diagram showing the overlap between the 5′ ss-associated factors and hits from our CRISPR screen. (E) STRING network analysis of the 17 shared factors. (F) eGFP signals from the eGFP-5′ ss-PAS reporter cell line treated with scramble or specific <t>sgRNAs</t> against the specified factors (related to Extended Data Fig. 1H ). (G) Quantified eGFP signals from the eGFP-5′ ss-PAS reporter cell line treated with scramble or specific sgRNAs against the specified factors.
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    (A) Workflow of a genome-wide CRISPR screen to identify candidate factors involved in 5′ ss-mediated gene repression. 293 Flp-In cells expressing the eGFP-5′ ss-PAS reporter are infected with a <t>lentiviral</t> <t>Brunello</t> genome-wide sgRNA library and selected with puromycin for 5 days. The selected cells are then treated with doxycycline for 3 days to induce eGFP expression. Following induction, the top 1% of high eGFP-expressing cells are sorted via FACS analysis. Genomic DNA is then extracted, followed by sgRNA barcode amplification and deep sequencing analysis. (B) Candidate genes identified by the CRISPR-Cas9 screen. Data analysis is performed using the MAGeCK algorithm to identify enriched sgRNA, and the genes are ranked by false discovery rate (FDR). Names of top candidates were labeled. (C) A schematic diagram for the RNA pull-down and mass spectrometry analysis. MBP-MS2, a fusion protein combining maltose-binding protein (MBP) and MS2, facilitating RNA capture and purification. 5′ ss: 5′ splicing site; WT: wild type; Mut: mutant. (D) A Venn diagram showing the overlap between the 5′ ss-associated factors and hits from our CRISPR screen. (E) STRING network analysis of the 17 shared factors. (F) eGFP signals from the eGFP-5′ ss-PAS reporter cell line treated with scramble or specific <t>sgRNAs</t> against the specified factors (related to Extended Data Fig. 1H ). (G) Quantified eGFP signals from the eGFP-5′ ss-PAS reporter cell line treated with scramble or specific sgRNAs against the specified factors.
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    (A) Workflow of a genome-wide CRISPR screen to identify candidate factors involved in 5′ ss-mediated gene repression. 293 Flp-In cells expressing the eGFP-5′ ss-PAS reporter are infected with a lentiviral Brunello genome-wide sgRNA library and selected with puromycin for 5 days. The selected cells are then treated with doxycycline for 3 days to induce eGFP expression. Following induction, the top 1% of high eGFP-expressing cells are sorted via FACS analysis. Genomic DNA is then extracted, followed by sgRNA barcode amplification and deep sequencing analysis. (B) Candidate genes identified by the CRISPR-Cas9 screen. Data analysis is performed using the MAGeCK algorithm to identify enriched sgRNA, and the genes are ranked by false discovery rate (FDR). Names of top candidates were labeled. (C) A schematic diagram for the RNA pull-down and mass spectrometry analysis. MBP-MS2, a fusion protein combining maltose-binding protein (MBP) and MS2, facilitating RNA capture and purification. 5′ ss: 5′ splicing site; WT: wild type; Mut: mutant. (D) A Venn diagram showing the overlap between the 5′ ss-associated factors and hits from our CRISPR screen. (E) STRING network analysis of the 17 shared factors. (F) eGFP signals from the eGFP-5′ ss-PAS reporter cell line treated with scramble or specific sgRNAs against the specified factors (related to Extended Data Fig. 1H ). (G) Quantified eGFP signals from the eGFP-5′ ss-PAS reporter cell line treated with scramble or specific sgRNAs against the specified factors.

    Journal: bioRxiv

    Article Title: LENG8 mediates RNA nuclear retention and degradation in eukaryotes

    doi: 10.1101/2025.08.14.670437

    Figure Lengend Snippet: (A) Workflow of a genome-wide CRISPR screen to identify candidate factors involved in 5′ ss-mediated gene repression. 293 Flp-In cells expressing the eGFP-5′ ss-PAS reporter are infected with a lentiviral Brunello genome-wide sgRNA library and selected with puromycin for 5 days. The selected cells are then treated with doxycycline for 3 days to induce eGFP expression. Following induction, the top 1% of high eGFP-expressing cells are sorted via FACS analysis. Genomic DNA is then extracted, followed by sgRNA barcode amplification and deep sequencing analysis. (B) Candidate genes identified by the CRISPR-Cas9 screen. Data analysis is performed using the MAGeCK algorithm to identify enriched sgRNA, and the genes are ranked by false discovery rate (FDR). Names of top candidates were labeled. (C) A schematic diagram for the RNA pull-down and mass spectrometry analysis. MBP-MS2, a fusion protein combining maltose-binding protein (MBP) and MS2, facilitating RNA capture and purification. 5′ ss: 5′ splicing site; WT: wild type; Mut: mutant. (D) A Venn diagram showing the overlap between the 5′ ss-associated factors and hits from our CRISPR screen. (E) STRING network analysis of the 17 shared factors. (F) eGFP signals from the eGFP-5′ ss-PAS reporter cell line treated with scramble or specific sgRNAs against the specified factors (related to Extended Data Fig. 1H ). (G) Quantified eGFP signals from the eGFP-5′ ss-PAS reporter cell line treated with scramble or specific sgRNAs against the specified factors.

    Article Snippet: The Human CRISPR Knockout Pooled Library (Brunello) targeting 19,114 genes with a total of 77,441 sgRNAs (4 sgRNAs per gene) was obtained from Addgene (Pooled Library #73179).

    Techniques: Genome Wide, CRISPR, Expressing, Infection, Amplification, Sequencing, Labeling, Mass Spectrometry, Binding Assay, Purification, Mutagenesis