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    Illumina Inc rna library
    In vitro reconstitution of CBP <t>RNA</t> binding A) CBP domains and RNA binding prediction (BindN). Non-binding (green) and binding residues (red). Magnified sequence shows predicted RBR in CBP-HAT wt . B-C) In vitro pull down of B) eRNA-Klf6 s and C) eRNA-Med13l s . D) Quantification of RNA-pulldown data in B-C. n=3. E) In vitro pull down of eRNA-Klf6 s . F) Quantification of RNA-pulldown data in B-C. n=3. G-H) RNA EMSA of eRNAs using CBP-HAT wt . G) eRNA-Mdm2 s ; H) eRNA-Med13l s . I-J) Competition binding RNA EMSAs. Binding of 2nM 32 -P radiolabelled eRNA-Mdm2 to CBP-HAT wt (2000nM) was competed with: I) 0–20nM unlabelled eRNA-Mdm2; J) 1nM, 10nM and 20nM un-labeled eRNA-Mdm2 (RNA), dsDNA or ssDNA with the same sequence. K) RNA EMSA using: K) CBP-HAT delta-loop and eRNA-Mdm2; L) CBP-HAT mutant-loop and eRNA-Med13l. RNA was titrated with 0–8000nM CBP-HAT. (*) CBP-HAT wt (2000nM). M) RBR mediates RNA binding to FL-CBP in vivo . <t>PAR-CLIP</t> for GFP-tagged CBP wt , CBP delta-loop or CBP mutant-loop in MEFs was followed by RT-qPCR. Control lncRNA Malat-1 was not identified by PARalyzer v1.1. P -values from two-tailed Student’s t-test: *P
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    In vitro reconstitution of CBP RNA binding A) CBP domains and RNA binding prediction (BindN). Non-binding (green) and binding residues (red). Magnified sequence shows predicted RBR in CBP-HAT wt . B-C) In vitro pull down of B) eRNA-Klf6 s and C) eRNA-Med13l s . D) Quantification of RNA-pulldown data in B-C. n=3. E) In vitro pull down of eRNA-Klf6 s . F) Quantification of RNA-pulldown data in B-C. n=3. G-H) RNA EMSA of eRNAs using CBP-HAT wt . G) eRNA-Mdm2 s ; H) eRNA-Med13l s . I-J) Competition binding RNA EMSAs. Binding of 2nM 32 -P radiolabelled eRNA-Mdm2 to CBP-HAT wt (2000nM) was competed with: I) 0–20nM unlabelled eRNA-Mdm2; J) 1nM, 10nM and 20nM un-labeled eRNA-Mdm2 (RNA), dsDNA or ssDNA with the same sequence. K) RNA EMSA using: K) CBP-HAT delta-loop and eRNA-Mdm2; L) CBP-HAT mutant-loop and eRNA-Med13l. RNA was titrated with 0–8000nM CBP-HAT. (*) CBP-HAT wt (2000nM). M) RBR mediates RNA binding to FL-CBP in vivo . PAR-CLIP for GFP-tagged CBP wt , CBP delta-loop or CBP mutant-loop in MEFs was followed by RT-qPCR. Control lncRNA Malat-1 was not identified by PARalyzer v1.1. P -values from two-tailed Student’s t-test: *P

    Journal: Cell

    Article Title: RNA binding to CBP stimulates histone acetylation and transcription

    doi: 10.1016/j.cell.2016.12.020

    Figure Lengend Snippet: In vitro reconstitution of CBP RNA binding A) CBP domains and RNA binding prediction (BindN). Non-binding (green) and binding residues (red). Magnified sequence shows predicted RBR in CBP-HAT wt . B-C) In vitro pull down of B) eRNA-Klf6 s and C) eRNA-Med13l s . D) Quantification of RNA-pulldown data in B-C. n=3. E) In vitro pull down of eRNA-Klf6 s . F) Quantification of RNA-pulldown data in B-C. n=3. G-H) RNA EMSA of eRNAs using CBP-HAT wt . G) eRNA-Mdm2 s ; H) eRNA-Med13l s . I-J) Competition binding RNA EMSAs. Binding of 2nM 32 -P radiolabelled eRNA-Mdm2 to CBP-HAT wt (2000nM) was competed with: I) 0–20nM unlabelled eRNA-Mdm2; J) 1nM, 10nM and 20nM un-labeled eRNA-Mdm2 (RNA), dsDNA or ssDNA with the same sequence. K) RNA EMSA using: K) CBP-HAT delta-loop and eRNA-Mdm2; L) CBP-HAT mutant-loop and eRNA-Med13l. RNA was titrated with 0–8000nM CBP-HAT. (*) CBP-HAT wt (2000nM). M) RBR mediates RNA binding to FL-CBP in vivo . PAR-CLIP for GFP-tagged CBP wt , CBP delta-loop or CBP mutant-loop in MEFs was followed by RT-qPCR. Control lncRNA Malat-1 was not identified by PARalyzer v1.1. P -values from two-tailed Student’s t-test: *P

    Article Snippet: Sequencing libraries for PAR-CLIP experiments were prepared using NEB next small RNA library set for Illumina sequencing (New England Biolabs) according to manufacturers instructions.

    Techniques: In Vitro, RNA Binding Assay, Binding Assay, Sequencing, HAT Assay, Labeling, Mutagenesis, In Vivo, Cross-linking Immunoprecipitation, Quantitative RT-PCR, Two Tailed Test

    CBP interacts with RNA in vivo A) Native RNA-IP of CBP. Top, RNA immunprecipitated with CBP. Bottom, CBP western blot. B) PAR-CLIP protocol. 4-Thiouridine (4-SU). C) CBP PAR-CLIP required 4-SU: top, autoradiography; bottom, CBP western blot.. D) Quantification of CBP PAR-CLIP. Error bars represent mean +/− s.e.m; n=4. E) CBP PAR-CLIP signal was sensitive to RNAse. 1× RNAse cocktail contained: RNAse A (0.01mU/ul) + RNase T1 (0.4mU/ul). F) Quantification of RNase titration. Error bars represent mean +/− s.e.m; n=4; P -values from two-tailed Student’s t-test: *P

    Journal: Cell

    Article Title: RNA binding to CBP stimulates histone acetylation and transcription

    doi: 10.1016/j.cell.2016.12.020

    Figure Lengend Snippet: CBP interacts with RNA in vivo A) Native RNA-IP of CBP. Top, RNA immunprecipitated with CBP. Bottom, CBP western blot. B) PAR-CLIP protocol. 4-Thiouridine (4-SU). C) CBP PAR-CLIP required 4-SU: top, autoradiography; bottom, CBP western blot.. D) Quantification of CBP PAR-CLIP. Error bars represent mean +/− s.e.m; n=4. E) CBP PAR-CLIP signal was sensitive to RNAse. 1× RNAse cocktail contained: RNAse A (0.01mU/ul) + RNase T1 (0.4mU/ul). F) Quantification of RNase titration. Error bars represent mean +/− s.e.m; n=4; P -values from two-tailed Student’s t-test: *P

    Article Snippet: Sequencing libraries for PAR-CLIP experiments were prepared using NEB next small RNA library set for Illumina sequencing (New England Biolabs) according to manufacturers instructions.

    Techniques: In Vivo, Western Blot, Cross-linking Immunoprecipitation, Autoradiography, Titration, Two Tailed Test