Project description:Estrogen Receptor alpha (ERα) is a key driver of most breast cancers, and it is the target of endocrine therapies used in the clinic to treat women with ERα positive (ER+) breast cancer. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used to pull down the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.
Project description:Genomic locations of Myc-tagged budding yeast Rif1 (including wilt-type and designer mutants) were analysed by ChIP-Seq. Mutants tested were rif1-7A and rif1-7E, in which Ser/Thr residues in the cluster of SQ/TQ sites were mutated to Ala or Glu, respectively. The performance of RIF1-9V5 was also tested.
Project description:We use ChIP-seq to identify the targets of Efg1 in Candida parapsilosis. We show that Efg1 binds to 502 promoter regions, including 70 potential transcription factors or regulatory proteins. Several of the transcription factors belong to networks that regulate biofilm development and white-opaque switching in C. albicans. Efg1 also binds to its own promoter. The binding site for C. parapsilosis Efg1 resembles that of orthologs in other fungi. Many Efg1 targets are probably also regulated by the Ndt80 transcription factor. Efg1 in C. parapsilosis was tagged in situ using a myc epitope. Examination of Efg1-myc DNA binding sites by ChIP-seq. Three immunoprecipitated samples were sequenced and three input samples (pre-immunoprecipitation) were sequenced as controls.
Project description:After finding a QTL hotspot in a fission yeast cross of strains 968xY0036 that was apparently driven by frame shift mutation in swc5 (swc5-fs), we have determined the genome-wide occupancy of the histone variant H2AZ (PhT1). Indeed swc5 has been shown to affect H2AZ occupancy. To test the impact of swc5-fs on H2A.Z deposition, we performed genome-wide chromatin immuno-precipitation of H2A.Z coupled with deep sequencing (ChIP-seq) in the two parental strains (968, Y0036) and in a swc5 deletion strain. We also assed the histone H3 occupancy as a positive control. We generated pht1-myc tagged strains to precipitate H2AZ with Myc antibody. Several negative controls have been performed, in particular pulldowns with the non tagged strains. Results show a reduced H2A.Z occupancy at the +1 histone in both Y0036 and swc5-deletion strains.
Project description:Myc is a master transcription factor that has been demonstrated to be required for embryonic stem cell (ESC) pluripotency, self-renewal, and inhibition of differentiation. Although recent works identified several Myc-targets in ESC the list of Myc binding sites is largely incomplete due to the low sensitivity and specificity of the antibodies available so far. To systematically identify Myc binding sites in mouse ESCs here we used a stringent streptavidin based genome-wide chromatin immunoprecipitation (ChIP-Seq) of a biotin-tagged Myc (Bio-Myc) as well as a ChIP-Seq of the Myc partner Max. This analysis identified 4273 Myc binding sites of which more than 85% co-occupied by Max, overlap with H3K4me3 positive promoters and active enhancers of transcriptional regulators, chromatin modifiers, and genes involved in stem cell self-renewing. The new sites identified were validated experimentally. This study provides a new Myc and Max binding reference in mouse ESCs. ChIP-seq of bio-Myc and Max in E14 and respective controls; Bio-Myc ChIP-seq was performed in a stable clone of E14 mouse embryonic stem cell expressing Biotin-tagged Myc; Mock, Max and IgG were performed in parental wt E14 mESC.
Project description:As part of a study on the function of Rap1 at HML and MAT in Saccharomyces cerevisiae, we performed ChIP-seq on myc-tagged Sir3 in cells with and without a Rap1 binding site mutation at the promoter.
Project description:ChIP-seq was performed to map the association of SPA-tagged DnaA across the Escherichia coli MG1655 chromosome during exponential phase growth in LB. As a control to remove background, ChIP-Seq was also performed on SPA-tagged AcpS, a protein that is not known to bind DNA.