guide rna grna sequences targeting brd4  (Addgene inc)

Journal: Science Advances

Article Title: Targeting BRD3 eradicates nuclear TYRO3-induced colorectal cancer metastasis

doi: 10.1126/sciadv.ade3422

Figure Lengend Snippet: ( A ) Representative Western blots and quantified result show SNAI1 in cells transfected with indicated plasmids in the presence (+) or absence (−) of MMP-2 inhibitor, ARP100 (1 μM). ( B ) Representative Western blots and quantified result show the levels of SNAI1 in HCT116 cells treated with (+) or without (−) BRD3 inhibitor, RVX-208 (10 μM). ( C and D ) Representative images and quantified results show BrdU + (C) and annexin V + /PI + (D) cells treated with or without BRD3 inhibitor. Scale bar, 50 μm. ( E ) Real-time reverse transcription–qRCR shows the expression of SNAI1. ( F ) Representative Western blot and quantified result show SNAI1 in ICD-TYRO3–overexpressed HCT116 cells with or without BRD3 knockout. ( G ) Representative Western blot and quantified result show global acetylation of histone H3 in HCT116 cells. ( H ) Potential target genes of BRD3 were analyzed by ChIP-qPCR. ( I ) Representative images show location of C-TYRO3, E-cadherin, and DAPI in colon organoids. Scale bar, 10 μm. ( J ) Representative images and quantified result of organoids ( n = 3, 30 organoids per subject) treated with or without inhibitors were shown. Scale bar, 100 μm. ( K ) Representative images show local invasion and metastasis of cancer cells with or without different drug treatments. Arrows indicate the invasive cancer. Scale bar: 100 μm (left column); 50 μm (middle and right columns). ( L ) Representative images show local invasion and metastasis of cancer cells with or without BRD3 (sgBRD3) or BRD4 (sgBRD4) knockout. Arrows indicate the invasive cancer. Scale bars, 100 μm (left) and 50 μm (right). * P < 0.05, ## P < 0.01, ### and *** P < 0.001.

Article Snippet: To generate CRISPR-Cas9–mediated BRD3 and BRD4 knockout cells, the predesigned and validated guide RNA (gRNA) sequences targeting BRD3 and BRD4 ( ) were synthesized and cloned into eSpCas9-2A-Neo vector that was customizedly designed and modified from eSpCas9-2A-Puro (PX459) V2.0 by GenScript ( www.genscript.com/ ).

Techniques: Western Blot, Transfection, Reverse Transcription, Expressing, Knock-Out, ChIP-qPCR

Journal: Nature cell biology

Article Title: Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity

doi: 10.1038/s41556-018-0086-3

Figure Lengend Snippet: ( a ) Gene set enrichment plot showing that genes associated with high H3K9ac and H3K27ac are enriched for two independently defined pluripotency gene sets: Muller Plurinet (genes involved in the protein-protein network shared by diverse pluripotent cell types ) and Wong ESC Core (genes coordinately upregulated in mouse and human ESCs ). Data are derived from a single ChIP-Seq experiment . P values are calculated based on 1000 permutations by the GSEA algorithm and was not adjusted for multiple comparisons. ( b ) 2i increases acetylation at key pluripotency genes. H3K27ac (left) and H3K9ac (right) at enhancer (enh) or promoters of indicated genes as assessed by ChIP-qPCR. ( c ) ChIP-seq meta profile for Brd4 binding in ESCs cultured in S/L or S/L+2i. The metaprofile is centered on the midpoint of all Brd4 ChIP-seq peaks. ( d ) Brd4 ChIP-qPCR illustrating Brd4 binding in ESCs cultured in S/L (left) or S/L+2i (right) treated with DMSO (vehicle) or 500 nM JQ1 for 24 h. ( b,d ) Bars represent mean of n=3 technical replicates from one IP.

Article Snippet: Previously described guide RNA (gRNA) sequences targeting Brd4 or a nongenic region on mouse chromosome 8 (ch8) were cloned into the pCas9 (BB)2A-GFP (pX458, Addgene plasmid number Plasmid #48138), as previously described .

Techniques: Derivative Assay, ChIP-sequencing, ChIP-qPCR, Binding Assay, Cell Culture

Journal: Nature cell biology

Article Title: Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity

doi: 10.1038/s41556-018-0086-3

Figure Lengend Snippet: ( a ) Quantification of colony formation assay of cells cultured in S/L (left) or S/L+2i (right) transfected with Cas9 and the indicated sgRNA against a nongenic region of chromosome 8 (ch8, control) or exon 3 (ex3) or exon 4 (ex4) of Brd4 . Dotted line represents average of ch8 controls. Each bar represents quantification of a single well of a six-well plate; transfected samples were seeded in duplicate. ( b ) Alkaline phosphatase staining of colonies formed from single cells of clonal ESC lines edited with sgRNA against chromosome 8 (ch8) or Brd4 exon 3 and cultured in S/L or S/L+2i. One representative well of a six-well plate is shown. ( c ) Quantification of colony formation assay shown in ( b ). ( d ) Western blot depicting Brd4 levels in ESCs expressing doxycycline (dox)-inducible hairpins against Renilla (shRen) or Brd4 (shBrd4). Cells were cultured with or without dox for 48 h prior to harvest. Actin is used as a loading control. Western blot was performed two independent times. ( e ) Brightfield images of ESCs expressing shBrd4-2 cultured for 48 h with or without doxycycline (dox). ( f ) Population doublings of cells cultured for 72 h in doxycycline relative to controls grown without dox. ( g ) Alkaline phosphatase staining of colonies formed from single cells expressing the indicated hairpins grown in the presence or absence of doxycycline (dox) and cultured in S/L or S/L+2i. One representative well of a six-well plate is shown. All bars represent mean ±SEM ( c ) or ±SD ( f ) of n=3 independent samples. ****, P < 0.0001 by 2-way ANOVA with Sidak’s multiple comparisons post test (shBrd4-1, P = 7.2e-10; shBrd4-2, P = 4.6e-8). Scale bar, 100 μm.

Article Snippet: Previously described guide RNA (gRNA) sequences targeting Brd4 or a nongenic region on mouse chromosome 8 (ch8) were cloned into the pCas9 (BB)2A-GFP (pX458, Addgene plasmid number Plasmid #48138), as previously described .

Techniques: Colony Assay, Cell Culture, Transfection, Control, Staining, Western Blot, Expressing

Journal: Nature cell biology

Article Title: Pluripotency transcription factors and Tet1/2 maintain Brd4-independent stem cell identity

doi: 10.1038/s41556-018-0086-3

Figure Lengend Snippet: ( a ) qRT-PCR on key pluripotency genes in ESCs cultured in S/L or S/L+2i treated with DMSO (veh) or 500 nM JQ1 for 24h. ( b ) qRT-PCR on key pluripotency genes in S/L or S/L+2i-cultured ESCs expressing dox-inducible hairpins against Renilla (shRen) or Brd4 (shBrd4) and treated with dox for 48 h. Shown are levels of Nanog and Esrrb in cells with dox relative to control S/L+shRen cells without dox. ( c ) Heat map shows expression of pluripotency associated genes measured by RNA-Seq in ESCs cultured in S/L or S/L+2i with vehicle (veh, DMSO) or 500 nM JQ1 for 72 h. Color scale represents Log 2 relative to mean expression level. ( d ) Nanog binding to key pluripotency loci in ESCs cultured in S/L or S/L+2i treated with DMSO or 500 nM JQ1 for 24h, assessed by ChIP-qPCR. Bar represents mean of n=3 technical replicates from one IP. ( e ) ATAC-Seq meta profile of chromatin accessibility in ESCs cultured in S/L (left) or S/L+2i (right) with DMSO or 500 nM JQ1. Data from 2 independent replicates are shown. ( f ) At baseline, 82% of Oct4, Sox2, Nanog (OSN) binding sites have an ATAC-Seq peak above background. OSN sites make up 60% (4,250/7,084) of the peaks that maintain accessibility despite JQ1 treatment in S/L+2i cultured ESCs but only 17% (93/545) of peaks lost in both conditions. ( g ) ChIP-seq meta profile for Med1 binding in ESCs cultured in S/L or S/L+2i treated 500 nM JQ1. ( h ) Box plot shows relative Med1 binding at n=635 genes that are downregulated > 2 fold, FDR < 5% by JQ1 in ESCs cultured in S/L but not in ESCs cultured in S/L+2i. Values from ChIP-Seq experiment shown in (g) with 1 ChIP per condition. Box, 25-75 th percentile; bar, median; whiskers, 5-95 th percentile. ****, P < 1e-15; ns, P = 0.27 by 2-way ANOVA with Tukey’s multiple comparisons post test. See . ( i ) Sites that gain Med1 binding in S/L+2i+JQ1 relative to S/L+JQ1 are mostly OSN binding sites. Data presented as mean ± SD of n=3 independent samples ( a , b ).

Article Snippet: Previously described guide RNA (gRNA) sequences targeting Brd4 or a nongenic region on mouse chromosome 8 (ch8) were cloned into the pCas9 (BB)2A-GFP (pX458, Addgene plasmid number Plasmid #48138), as previously described .

Techniques: Quantitative RT-PCR, Cell Culture, Expressing, Control, RNA Sequencing, Binding Assay, ChIP-qPCR, ChIP-sequencing