human gecko lentiviral sgrna library  (Addgene inc)

Journal: eLife

Article Title: Netrin signaling mediates survival of dormant epithelial ovarian cancer cells

doi: 10.7554/eLife.91766

Figure Lengend Snippet: ( A ) TCGA RNA-seq data for HGSOC patients (TCGA PanCancer Atlas study) was used to identify high Netrin-1 or –3 and low Netrin-1 or –3 expressing patients (high expressing are above z-score 1.2). Overall survival was used to construct Kaplan-Meier plots and survival was compared using a logrank test. ( B ) OVCAR8 cells were stably transduced with lentiviral constructs to overexpress epitope tagged Netrin-1 or –3. Western blotting for Netrins, Myc-tags, and Actin were used to determine relative expression levels of both Netrins in these cell populations and vector controls. ( C ) Control and Netrin overexpressing cells were transferred to suspension culture conditions to form spheroids, and replated to assay for viability. Mean viability and standard deviation is shown for each. One-way anova was used to compare survival (* p<0.05, *** p<0.001). ( D ) Reattached spheroids were fixed and stained with Crystal Violet to examine size and abundance in control and Netrin-1 or –3 overexpression. Figure 7—source data 1. Original files for western blot analysis in (Myc, Netrin-1, Netrin-3, Actin). Figure 7—source data 2. PDFs containing annotation of original western blot analysis in (Myc, Netrin-1, Netrin-3, Actin). Figure 7—source data 3. Numerical data used for graphs in .

Article Snippet: Recombinant DNA reagent , Human GeCKO Lentiviral sgRNA library V2LentiGuide Puro , Addgene , #1000000049 , Human whole genome CRISPR library.

Techniques: RNA Sequencing, Expressing, Construct, Stable Transfection, Transduction, Western Blot, Plasmid Preparation, Control, Suspension, Standard Deviation, Staining, Over Expression

Journal: eLife

Article Title: Netrin signaling mediates survival of dormant epithelial ovarian cancer cells

doi: 10.7554/eLife.91766

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , Human GeCKO Lentiviral sgRNA library V2LentiGuide Puro , Addgene , #1000000049 , Human whole genome CRISPR library.

Techniques: Western Blot, Plasmid Preparation, Recombinant, CRISPR, Expressing, Selection, Sequencing, Gel Extraction, DNA Extraction, cDNA Synthesis, SYBR Green Assay, Protease Inhibitor, Software, Illumina Sequencing, Immunohistochemistry, Staining, Knock-Out, Kinase Assay, Construct, Over Expression, Amplification, Cloning, Control

Journal: bioRxiv

Article Title: A YIPF5-GOT1A/B complex directs a transcription-independent function of ATF6 in ER export

doi: 10.1101/2023.12.12.569033

Figure Lengend Snippet: a) Schematic of pharmacogenetic screens to identify factors that modulate the effect of secretion inhibitors. b) General procedure of CRISPR activation and CRISPR knock-out (KO) screening approaches. MCF10A cells were infected with the genome-wide lentiviral SAM (synergistic activation mediator) or GeCKO v2 sgRNA library. After selection with Brefeldin A (BFA) or FLI-06, the sgRNA distribution was determined by NGS (next generation sequencing) and analyzed using the RSA (redundant siRNA activity) algorithm in comparison with unselected control cells. c) CRISPR activation screen for Brefeldin A (BFA, 100 nM) resistance in clonal MCF10A cells using the SAM system. Displayed is the RSA analysis for sgRNA enrichment from two MCF10A-SAM clones (#9 and #28). Each dot represents one gene, gene names of selected hits are shown. d-e ) Validation of GBF1 as BFA target. qPCR expression analysis ( d ) of GBF1 and IncuCyte growth curve analysis upon BFA treatment ( e ) in MCF10A-SAM cells stably expressing sgGBF1 or non-targeting (NT) sgRNAs. Displayed is the confluency over time. f ) Principle to identify genetic perturbations that confer resistance or sensitivity to secretion inhibitors by sgRNA enrichment or depletion, respectively. g ) CRISPR a screen for FLI-06 (10 µM) resistance in clonal MCF10A cells (#9 and #28) using the SAM system. RSA analysis for both clones was performed for sgRNA enrichment (left) or sgRNA depletion (right). Each dot represents one gene. h ) CRISPR-KO screen for FLI-06 (10 µM) resistance in human BJ-Tert cells using the GeCKO sgRNA library. RSA analysis was performed for sgRNA enrichment. Each dot represents one gene. i-n ) Validation of GOLT1A , GOLT1B and YIPF5 as mediators of resistance/sensitivity to FLI-06. qPCR expression analysis ( i ) of GOLT1A and IncuCyte growth curve analysis upon FLI-06 (10 µM) treatment ( j ) in MCF10A-SAM clone #9 stably expressing sgGOLT1A or non-targeting sgRNAs. qPCR expression analysis ( k ) of GOLT1B and IncuCyte growth curve analysis upon FLI-06 treatment ( l ) in MCF10A cells stably expressing shGOLT1B or shRenilla as control. m ) Immunoblotting analysis with total protein as loading control and n ) IncuCyte growth curve analysis upon FLI-06 treatment in YIPF5 KO and YIPF5 or empty vector re-expressing MCF10A cells. For qPCR, B2M was used as housekeeping gene. Data are shown as mean of three biological replicates and error bars represent SEM ( d, e, i, j, k, l, n ). Statistical testing: one-way-ANOVA test with Tukey HSD post-hoc test ( d, e, i, j, l, n ) and unpaired two-sided Welch t-test ( k ).

Article Snippet: The genome-wide human GeCKO lentiviral sgRNA library v2 (kind gift from Feng Zhang, Addgene #1000000048), consisting of the 2 pools A and B, was amplified as previously described ( ) and the balanced library distribution was verified by NGS for both of them.

Techniques: CRISPR, Activation Assay, Knock-Out, Infection, Genome Wide, Selection, Next-Generation Sequencing, Activity Assay, Comparison, Control, Clone Assay, Biomarker Discovery, Expressing, Stable Transfection, Western Blot, Plasmid Preparation