monarch spin pcr and dna cleanup kit  (New England Biolabs)

Journal: Oncology Reports

Article Title: Perturbation of BRD4 and p300 activity suppresses super enhancer-driven KLF6 expression in renal carcinoma

doi: 10.3892/or.2026.9118

Figure Lengend Snippet: Changes in H3K27ac enrichment at the KLF6 enhancer regions following BRD4 and p300 inhibition. (A) Schematic representation of three constituent enhancers located in proximity to the KLF6 locus. (B) Pharmacological inhibition of BRD4 using JQ1 decreased the H3K27ac signal compared with the vehicle control. (C) Pharmacological inhibition of p300 using A-485 decreased the H3K27ac signal compared with the vehicle control. IgG ChIP-quantitative PCR showed minimal enrichment at all KLF6 enhancer regions following treatment with JQ1 and A-485, as well as their respective vehicle controls, confirming low background signal. P-values were determined using unpaired t-test with Welch's correction. *P<0.05. ns, not significant; H3K27ac, acetylation at lysine 27 of histone H3; KLF6, Kruppel-like factor 6; BRD4, bromodomain-containing 4; iSE, Clustered Regularly Interspaced Short Palindromic Repeats Interference-targeted super enhancer; ChIP, chromatin immunoprecipitation.

Article Snippet: To reverse cross-links and elute bound DNA, 100 μl elution buffer was added and samples were incubated at 65°C with shaking at 1,000 rpm for 3 h. DNA was purified using the Monarch PCR & DNA Cleanup kit (New England BioLabs, Inc.) according to the manufacturer's protocol. qPCR was performed by adding the purified, de-crosslinked DNA to a reaction mix containing 2X PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Inc.) and 10 μM forward and reverse ChIP-qPCR iSE_1, ChIP qPCR iSE_2 and ChIP qPCR iSE_3 primers ( ).

Techniques: Inhibition, Control, Real-time Polymerase Chain Reaction, CRISPR, Chromatin Immunoprecipitation

Journal: Oncology Reports

Article Title: Perturbation of BRD4 and p300 activity suppresses super enhancer-driven KLF6 expression in renal carcinoma

doi: 10.3892/or.2026.9118

Figure Lengend Snippet: CRISPR-mediated deacetylation of SE_1 decreases KLF6 expression. (A) Western blot analysis confirmed stable dCas9-HDAC3 expression in 786-M1A cells transduced with single guide RNAs. (B) qPCR analysis showed reduced KLF6 expression in iSE_1-transduced cells compared with NTC cells. P-values were determined by one-way ANOVA followed by Dunnett's multiple-comparison post hoc test. (C) ChIP-qPCR showed decreased H3K27ac enrichment at SE_1 in iSE_1-transduced cells compared with NTC cells. IgG ChIP-qPCR showed minimal enrichment at SE_1 in both NTC and iSE_1 transduced cells, confirming low background signal and antibody specificity. P-values were determined using an unpaired t-test with Welch's correction. *P<0.05, **P<0.005. dCas9-HDAC3, dead Cas9-histone deacetylase 3; qPCR, quantitative PCR; KLF6, Kruppel-like factor 6; iSE, CRISPRi-targeted super enhancer; NTC, non-targeting control; ChIP, chromatin immunoprecipitation; H3K27ac, acetylation at lysine 27 of histone H3; ns, not significant.

Article Snippet: To reverse cross-links and elute bound DNA, 100 μl elution buffer was added and samples were incubated at 65°C with shaking at 1,000 rpm for 3 h. DNA was purified using the Monarch PCR & DNA Cleanup kit (New England BioLabs, Inc.) according to the manufacturer's protocol. qPCR was performed by adding the purified, de-crosslinked DNA to a reaction mix containing 2X PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Inc.) and 10 μM forward and reverse ChIP-qPCR iSE_1, ChIP qPCR iSE_2 and ChIP qPCR iSE_3 primers ( ).

Techniques: CRISPR, Expressing, Western Blot, Transduction, Comparison, ChIP-qPCR, Histone Deacetylase Assay, Real-time Polymerase Chain Reaction, Control, Chromatin Immunoprecipitation