Journal: Cell reports

Article Title: Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells

doi: 10.1016/j.celrep.2022.111226

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pXR001 (NLS-RfxCas13d-NLS-HA-T2A-EGFP) , ( ) , Addgene #109049.

Techniques: Virus, Recombinant, Staining, Transfection, Electron Microscopy, Protease Inhibitor, Blocking Assay, Viability Assay, Plasmid Preparation, Reverse Transcription, Bicinchoninic Acid Protein Assay, Magnetic Beads, shRNA, Cloning, Software, Generated, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: The chromatin associated Sin3B is a critical regulator of DNA damage repair through the engagement of the Non-Homologous End Joining

doi: 10.1101/2022.03.09.483624

Figure Lengend Snippet: A. Sin3B +/+ and Sin3B -/- T98G cells were synchronized in different phases of the cell cycle before being treated with 25 ug/mL of cisplatin for 12 followed by Annexin V staining followed by flow cytometry analysis. Annexin V positive cells were then quantified. Error bars represent SD; n = 3. Significance was determined by two-way ANOVA followed by a Tukey’s test. **** P <0.0001. B. Cells were treated with 25 ug/mL of cisplatin for 4 h. Cells were then fixed, stained for 53BP1 and imaged. Representative images of the data quantified in 2C. C. Quantification of cells with 53BP1 foci. Error bars represent SD; n = 3. Significance was determined by two-way ANOVA followed by a Sidak’s test. *** P <0.001. D. T98G cells containing the EJ5-GFP reporter were analyzed for GFP positivity, 72 hours after nuclease induction. The percentage of GFP + cells were determined for each individual condition and subsequently normalized to transfection efficiency. Error bars represent SD; n = 3. Significance was determined by Student’s t-test. ** P <0.01. E. T98G Sin3B +/+ and Sin3B -/- TLR cell lines were created and then infected with lentivirus containing SceI and the template for eGFP reconstitution. After 72 hours, percentage of GFP + cells were determined by flow cytometry for each individual condition and subsequently normalized to infection efficiency. Error bars represent SD; n = 3. Significance was determined by Student’s t-test. * P <0.05. F. Similar to E, RFP + cells were determined by flow cytometry. Error bars represent SD; n = 3. Significance was determined by Student’s t-test. **** P <0.0001. G. Sin3B +/+ and Sin3B -/- T98G cells were treated with 25 ug/mL of cisplatin for 12 h and 10 nM NU7441. Cells were then subjected to Annexin V staining followed by flow cytometry analysis. Annexin V positive cells were then quantified. Error bars represent SD; n = 3. Significance was determined by two-way ANOVA followed by a Tukey’s test. **** P <0.0001.

Article Snippet: T98G Sin3B +/+ and Sin3B -/- TLR cell lines were created and then infected with lentivirus containing SceI and the template for eGFP reconstitution (Addgene #32627), after 72 hours, flow cytometric analysis was performed.

Techniques: Staining, Flow Cytometry, Transfection, Infection

Journal: Cell reports

Article Title: Negative autoregulation mitigates collateral RNase activity of repeat-targeting CRISPR-Cas13d in mammalian cells

doi: 10.1016/j.celrep.2022.111226

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pXR002 (NLS-dRfxCas13d-NLS-HA-T2A-EGFP) , ( ) , Addgene #109050.

Techniques: Virus, Recombinant, Staining, Transfection, Electron Microscopy, Protease Inhibitor, Blocking Assay, Viability Assay, Plasmid Preparation, Reverse Transcription, Bicinchoninic Acid Protein Assay, Magnetic Beads, shRNA, Cloning, Software, Generated, Fluorescence, Microscopy