cut&run assay kit cst #86652  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: A genome-wide single-cell 3D genome atlas of lung cancer progression

doi: 10.1101/2023.07.23.550157

Figure Lengend Snippet: a, Diagram illustration of the prediction procedure that identified Rnf2 as a candidate. b, Western blot analysis of Rnf2 protein levels following Rnf2 knockdown with three independent hairpins in the KP cell line. Control cells were constructed with a non-targeting control shRNA sequence (shNTC). HSP90 is loading control. c, Cell viability (mean ± SD, normalized to mean of shNTC) following knockdown with three Rnf2 shRNAs, n = 3 replicates per hairpin. p values from two-sided two-sample t-tests are shown. d, Continuous assessment of cell proliferation (CellCyte) reveals that cell growth rate is reduced following Rnf2 knockdown (shRnf2-1). Error bars represent 95% confidence intervals. e, scA/B score changes from shRnf2 to shNTC versus those from AdenomaG to LUAD show a significant positive correlation, n = 473 target genomic regions. Black lines are fitted regression lines. Pearson correlation coefficients and p values are shown. f, Rnf2 peak densities in A (n = 209) and B (n = 264) compartments in shNTC. The horizontal lines of each box from top to bottom represent the 75th percentile, median, and 25th percentile. Whiskers extend to the non-outlier maximum and non- outlier minimum. Outliers are defined as values at least 1.5 times interquartile range away from the top or bottom of the box. p value of two-sided Wilcoxon rank-sum test is shown.

Article Snippet: Antibodies were used as listed below, with 0.5 ug of antibody per reaction: IgG Control antibody – CUTANA Kit Rabbit IgG CUT&RUN Negative Control Antibody RNF2 antibody – CST RING1B (D22F2) XP® Rabbit mAb #5694 H3K4me3 antibody – Epicypher Rabbit Polyclonal H3K4me3 13-0041 H3K27me3 antibody – CST Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733 H2AK119ub antibody – CST Ubiquityl-Histone H2A (Lys119) (D27C4) Rabbit mAb #8240 BMI-1 antibody – Active Motif BMI-1 antibody (mAb) 39993 RNA Pol II p-ser5 – Abcam Anti-RNA polymerase II phosphor-S5 EPR19015

Techniques: Western Blot, Knockdown, Control, Construct, shRNA, Sequencing

Journal: bioRxiv

Article Title: A genome-wide single-cell 3D genome atlas of lung cancer progression

doi: 10.1101/2023.07.23.550157

Figure Lengend Snippet: a, CUT&RUN read density heatmaps of Rnf2, H3K4me3, H3K27me3, RNA polymerase II with phosphorylated S5 modification, H2AK119ub, and BMI1 in shNTC KP cells. Rnf2 peak regions [-5k, +5k] of all target genomic regions are shown and are categorized as active (H3K4me3+, H3K27me3-), bivalent (H3K4me3+, H3K27me3+), repressive (H3K4me3-, H3K27me3+), or other (H3K4me3-, H3K27me3-) based on chromatin marks. b, scA/B score changes from shRnf2 to shNTC versus those from AdenomaG to LUAD show a stronger positive correlation using target genomic regions with only active Rnf2 peaks, n = 113 target genomic regions. c , Western blot analysis of Rnf2 protein levels following rescue of shRNA knockdown (shRnf2-3) with stable transduction of Rnf2 WT, ubiquitin-ligase dead Rnf2 I53S mutant, or empty vector (EV) control. HSP90 is loading control. d , scA/B score changes from shRnf2-3 to shRnf2-3+WT Rnf2 (left) and to shRnf2-3+I53S Rnf2 (right) versus those from AdenomaG to LUAD show a stronger positive correlation with expression of a catalytically dead mutant of Rnf2, n = 473 target genomic regions. In b, d, the black lines are fitted regression lines. Pearson correlation coefficients and p values are shown.

Article Snippet: Antibodies were used as listed below, with 0.5 ug of antibody per reaction: IgG Control antibody – CUTANA Kit Rabbit IgG CUT&RUN Negative Control Antibody RNF2 antibody – CST RING1B (D22F2) XP® Rabbit mAb #5694 H3K4me3 antibody – Epicypher Rabbit Polyclonal H3K4me3 13-0041 H3K27me3 antibody – CST Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733 H2AK119ub antibody – CST Ubiquityl-Histone H2A (Lys119) (D27C4) Rabbit mAb #8240 BMI-1 antibody – Active Motif BMI-1 antibody (mAb) 39993 RNA Pol II p-ser5 – Abcam Anti-RNA polymerase II phosphor-S5 EPR19015

Techniques: Modification, Western Blot, shRNA, Knockdown, Transduction, Ubiquitin Proteomics, Mutagenesis, Plasmid Preparation, Control, Expressing

Journal: bioRxiv

Article Title: A genome-wide single-cell 3D genome atlas of lung cancer progression

doi: 10.1101/2023.07.23.550157

Figure Lengend Snippet: During lung cancer progression from AT2 to preinvasive adenoma to LUAD, a 3D genomic structural bottleneck exists at the preinvasive adenoma stage, indicating an early stringent selection of the 3D genome conformation. The structural bottleneck manifests in 3D genome conformation heterogeneity, compaction, long-range intermixing, A-B compartment polarization, Rabl configuration, etc. Single-cell 3D genome organization encodes different cancer cell states and predicts cancer progression drivers and genetic vulnerabilities. Finally, Rnf2 – through a ubiquitin ligase-independent function – partially reorganizes A-B compartmentalization during the adenoma-to-LUAD transition. The cartoons were created with BioRender.com.

Article Snippet: Antibodies were used as listed below, with 0.5 ug of antibody per reaction: IgG Control antibody – CUTANA Kit Rabbit IgG CUT&RUN Negative Control Antibody RNF2 antibody – CST RING1B (D22F2) XP® Rabbit mAb #5694 H3K4me3 antibody – Epicypher Rabbit Polyclonal H3K4me3 13-0041 H3K27me3 antibody – CST Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733 H2AK119ub antibody – CST Ubiquityl-Histone H2A (Lys119) (D27C4) Rabbit mAb #8240 BMI-1 antibody – Active Motif BMI-1 antibody (mAb) 39993 RNA Pol II p-ser5 – Abcam Anti-RNA polymerase II phosphor-S5 EPR19015

Techniques: Selection, Ubiquitin Proteomics