length human brd4 protein  (Addgene inc)

Journal: Nature methods

Article Title: Accelerated Chromatin Biochemistry Using DNA-Barcoded Nucleosome Libraries

doi: 10.1038/nmeth.3022

Figure Lengend Snippet: Preparation of DNL and its use in in vitro ChIP-Seq experiments. ( a ) Modified histone variants prepared by protein semi-synthesis are assembled with the respective barcoded DNA into a barcoded nucleosome (‘NUC’) library (‘DNL’). After biochemical assays with a writer, reader or nuclear extract, the binders and reaction products are isolated by affinity- or immunoprecipitation, followed by DNA experiment multiplexing. NUC identity and abundance is analyzed by next generation sequencing (NGS). ( b ) Combinations of histone modifications (‘mod’) selected for the first version of the library (‘DNL-1’). Unmodified (‘–mod’) or modified H3 proteins (vertical axis) were combined with otherwise unmodified histones (‘–mod’), H2AK119ub, H2BK120ub, or mono-/hyperacetylated H4 (horizontal axis). Additionally, a NUC bearing H2BK120ub and H4Kac 5 was prepared. Asterisk: this variant was employed in the Brd4 experiment . ( c ) Analysis of the combined DNL-1 by native gel electrophoresis and ethidium bromide (EtBr) DNA staining. The bands from NUCs containing combinations of unmodified, Kac or Kme3 histones overlap, whereas the shifted fainter upper band represents NUCs containing ubiquitylated H2A or H2B. For details on modified histones, DNA preparation, NUC assembly and NGS, see .

Article Snippet: pFlag-CMV2-Brd4 (1–1362) encoding the cDNA of full-length human Brd4 protein was obtained from Addgene (plasmid #22304).

Techniques: In Vitro, ChIP-sequencing, Modification, Isolation, Immunoprecipitation, Multiplexing, Next-Generation Sequencing, Variant Assay, Nucleic Acid Electrophoresis, Staining

Journal: Nature methods

Article Title: Accelerated Chromatin Biochemistry Using DNA-Barcoded Nucleosome Libraries

doi: 10.1038/nmeth.3022

Figure Lengend Snippet: Profiling the substrate specificity of histone mark readers. ( a ) Left: Model showing bivalent nucleosomal recognition behavior of BPTF. Right: Immobilized GST-tagged BPTF-PHD (gray) and BPTF-PHD-BD (blue) were incubated with DNL-1, followed by DNA isolation and experiment multiplexing. The resulting DNA was analyzed by next generation sequencing, and the raw sequencing reads were normalized against input and the H3K4me3-containing variant, set as 1 (red asterisks). Values (mean ± SD, n=3) for the respective NUC variants are plotted as a 3D bar graph using the same grid as shown in . ( b ) Left: Model depicting potential binding modes of p300. Right: Library incubation of resin-bound GST-tagged p300-BD (gray) and p300-BD-PHD (blue). All subsequent steps were performed as in ( a ). Internal normalization: H4Kac 5 variant, set as 1 (red asterisks). Values are shown as (mean ± SD, n=3). ( c ) Left: Model depicting potential interaction between Brd4 and acetylated NUC. Right: Library incubation of resin-bound Flag-Brd4-BD1-BD2. All subsequent steps were performed as in ( a ) except for pulldown with an anti-Flag antibody. Internal normalization: H4Kac 5 variant, set as 1 (red asterisk). Experiment was performed in duplicate (replicate shown in ). See Source Data Table 1 for input-normalized sequencing reads.

Article Snippet: pFlag-CMV2-Brd4 (1–1362) encoding the cDNA of full-length human Brd4 protein was obtained from Addgene (plasmid #22304).

Techniques: Incubation, DNA Extraction, Multiplexing, Next-Generation Sequencing, Sequencing, Variant Assay, Binding Assay