Project description:A strain harboring two copies of RAP1 is used for a competition-ChIP experiment. One copy of RAP1 is expressed from the endogenous RAP1 promoter and a c-terminal 3X FLAG epitiope tag and the other is expressed from a weakened Galactose inducible promoter and a c-terminal 9X MYC tag. Following induction by 2% galactose Rap1-Myc and Rap1-Flag levels are determined genome wide using ChIP-chip.
Project description:On of the H3.3 genes (H3F3B) was C-terminally tagged with a recombination-induced tag exchange (RITE) tag in human RPE1-hTERT cells to follow old (V5 tag) and new (FLAG tag) H3.3 and measure the dynamics of H3.3. New H3.3 was induced by activating Cre recombinase with 4-hydrotamoxifen (4OHT). ChIP-seq was done on old H3.3 and new H3.3 8h and 24h after 4OHT treatment.
Project description:To identify Sp7’s molecular interactions in vivo, we used gene-targeting strategies to generate an Sp7-Biotin-3xFLAG knock-in mouse (Sp7-BioFL mouse). This approach appends a biotin (Bio) recognition motif and three copies of the FLAG (FL) epitope at the C-terminus of the Sp7 protein. To validate this knockin system, we compared ChIP-seq results in the MC3T3E1 osteoblast cell line, introducing Sp7 forms that were epitope-tagged at either the N- (N-terminal FLAG tag; MC3_FLAG-Sp7) or C-terminus (C-terminal Biotin-FLAG tag, as in the in vivo targeted allele; MC3_Sp7-BioFLAG). These data show an extensive overlap, suggesting that FLAG tagging at different positions gives comparable outcomes. Second, we examined ChIP on wild-type calvarial primary osteoblasts (POB) using an anti-Sp7 antibody (POB_Sp7Ab-ChIP). The majority of Sp7-Ab ChIP-seq peaks (95%) overlapped with the larger set identified by Sp7-BioFL ChIP-seq.
Project description:A strain harboring two copies of RAP1 is used for a competition-ChIP experiment. One copy of RAP1 is expressed from the endogenous RAP1 promoter and a c-terminal 3X FLAG epitiope tag and the other is expressed from a weakened Galactose inducible promoter and a c-terminal 9X MYC tag. Following induction by 2% galactose Rap1-Myc and Rap1-Flag levels are determined genome wide using ChIP-chip.
Project description:A strain harboring two copies of RAP1 is used for a competition-ChIP experiment. One copy of RAP1 is expressed from the endogenous RAP1 promoter and a c-terminal 3X FLAG epitiope tag and the other is expressed from a weakened Galactose inducible promoter and a c-terminal 9X MYC tag. Following induction by 2% galactose Rap1-Myc and Rap1-Flag levels are determined genome wide using ChIP-chip. Time Course ChIP-ChIP experiment, Rap1-Flag IP and Rap1-Myc IP. 10 Time Points (0,10,20,30,40,50,60,90,120,150 Minutes) 2 Biological Replicates. Total Rap1 Occupancy at times 0 and 60 minutes in the time course; 2 Biological Replicates. mRNA expression levels at times 0 and 60 minutes in the time course; 2 biological Replicates.
Project description:To investigate the genomic localization of NCoR/HDAC3/PGC1β complex and the enhancer/promoter activity in the regulation of osteoclast differentiation, we used NCoR KO or PGC1β KO bone marrow cells, non-coding RNA Dancr/Rnu12 siRNA knockdown bone marrow cells and FLAG-tagged PGC1β RNA recognition motif deletion mutant expressed RAW 264.7 cells. We then performed chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for H3K27ac, NCoR, HDAC3, PGC1β, p65, Fosl2, PU.1 and FLAG-tag.
Project description:We used insertional ChIP (iChIP) based approach to identify proteins that bind to mouse immunoglobulin switch (S) region. To perform iChIP, we inserted an 8X-repeat of the LexA-binding element (LexA-BE) downstream of the Sα region in CH12F3-2A cells, a mouse B-cell line. The DNA-binding domain and dimerization domain of the LexA protein fused with a FLAG tag and a nuclear localization signal (NLS) was expressed in WT (FNLDD-alone) or Sα-engineered (FNLDD-Sα-LexA) CH12F3-2A cells. The resultant cells were subjected to stable isotope labeling with amino acids in cell culture (SILAC), stimulated with CIT (CD40L, IL4, and TGFβ), immunoprecipitated with an anti-FLAG antibody, and subjected to LC-MS/MS analysis. The identities of the deposited data are as follows: F: WT (FNLDD-alone); D: Sα-engineered (FNLDD-Sα-LexA).
Project description:We report the results of ChIP-Seq experiment investigating the Rrf2 family transciptional regulator RsrR (putatively name Redox sensitive response regulator). The experiment utilized an in trans copy of rsrR with an N-terminal 3xFlag tag encoded on the plasmid pMS82 (containing a phiBT1 integration site). The experiment utilized beads associated with anti-flag antibodies, specific for the Flag tagged protein and a WT host strain. We report >600 target binding sites for the RsrR regulon encompassing a broad range of functional targets with specific reference to those associate with NADPH and NADH metabolism and synthesis.
Project description:We report the results of ChIP-Seq experiment investigating the MtrA TCS response regulator. The experiment utilised an in trans copy of mtrA with an N-terminal 3xFlag tag encoded on the plasmid pMS82 (containing a phiBT1 integration site). The experiment utilised beads associated with anti-flag antibodies, specific for the Flag tagged protein. We report important targets involved in cell division and salt stress with key findings supporting the hypothesis that MtrA is essential. Samples were taken over a time course of 10h, 12h, 14h, 16h, 18h and 20h.
Project description:dFOXO targets in adult Drosophila melanogaster females, and the effect of insulin signalling and stress on binding. The experimets determined the binding locations of dFOXO in the whole adult female fly using ChIP-chip. The protocol was validated using mock conditions: pre-immune serum or IP on chromatin from foxo null flies. The response of this binding to stress induced by treatment of flies with paraquat or by their exposure to starvation, as well as the response to an insulin-signalling-reducing genetic manipulation (over-expression of dominant negative form of the insulin receptor), was determined.