X-ChIP
X-ChIP
X-ChIP, crosslinked chromatin followed by immunoprecipitation, is a foundational technique in chromatin research. With the advent of NGS this simple technique, now called X-ChIP-seq, is able produce high-resolution results. The method involves cross-linking with 1% formaldehyde for 10 min at room temperature. The cells are washed and resuspended in Lysis buffer. After MNase digestion the chromatin is solubilized by brief sonication and then subjected to chromatin immunoprecipitation.
Pros:
- Single base resolution
Cons:
- Sonication may introduce sequence bias 1
- Captures protein–DNA interactions regardless of duration 2
- X-ChIP: Breiling A., Turner B. M., Bianchi M. E. and Orlando V. (2001) General transcription factors bind promoters repressed by Polycomb group proteins. Nature 412: 651-655
- X-ChIP: Negre N. and Cavalli G. (2001) Finding Downstream Target DNAs for Chromatin Proteins by X-CHIP in Drosophila. Mapping Protein/DNA Interactions by Cross-Linking
- X-ChIP-Seq: Skene P. J. and Henikoff S. (2015) A simple method for generating high-resolution maps of genome-wide protein binding. Elife 4: e09225
- 1. Teytelman L., Ozaydin B., Zill O., Lefrancois P., Snyder M., et al. (2009) Impact of chromatin structures on DNA processing for genomic analyses. PLoS One 4: e6700
- 2. Zentner G. E., Kasinathan S., Xin B., Rohs R. and Henikoff S. (2015) ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo. Nat Commun 6: 8733
- Schmidt S. V., Krebs W., Ulas T., Xue J., Bassler K., et al. (2016) The transcriptional regulator network of human inflammatory macrophages is defined by open chromatin. Cell Res 26: 151-170
- Elsasser S. J., Noh K. M., Diaz N., Allis C. D. and Banaszynski L. A. (2015) Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells. Nature 522: 240-244