Research stay in the US: ChIP-seq

I’ve been at Rutgers, the State University of New Jersey, for a couple of weeks now, learning how to do chromatin immunoprecipitation (ChIP, which I’ve discussed here).  This procedure, developed in the 80s, is a very powerful  technique for monitoring the association of proteins of interest to DNA in vivo. With the advent of new sequencing technologies, you now can identify all regions in a particular genome where a protein binds, by performing deep-sequencing to the immunoprecipitated DNA (ChIP-Seq, see “Analyzing the genome-wide chromatin landscape“).

That’s exactly what we intend to do.

Overview of the ChIP-seq process (Figure from Leleu et al, 2010) 2

We are doing the ChIP assays from purified nuclei, which according to our collaborator gives better results. In fact, the only ChIP-seq that has been published for Neurospora, used purified nuclei1. In that paper, the authors used ChIP-seq to identify the targets of the White Collar Complex (WCC), a transcriptional assembly composed of two proteins (WC-1 and WC-2), in response to a short light pulse. The WCC is responsible for most blue-light responses in this fungus and is also a core element of the Neurospora circadian clock (see here).

Doing the nuclei prep (which involves a sucrose gradient) takes a long time, particularly when you have several samples (a consequence of circadian research) but so do all the following steps. In any case, it has been really interesting to learn this powerful technique, which I’m sure will of great use to my labmates once I go back home.

Another thing that I’ll be learning while I’m in the States, is the bioinformatic part of the ChIP-Seq: how to analyze the data, map the reads, make peak calls, etc. All that is involved from generating the raw data to ultimately deciphering all the information derived from the ChIP-seq.

Viewing the data (From Leleu et al., 2010) 2

For this I will move to Philly, but I still have a couple of weeks here in Rutgers in which I’ll do more ChIP assays and, very importantly, validate them, before firing up the sequencers.  Usually this is done by testing through qPCR, positive and negative controls, in order to assess the specificity of the antibody, background levels, etc.

Well, I’ll be positing every now and then about how the research stay is going. At least, I’ve secured a place to stay in Philly, thanks to @aanaqvi!

Stay tuned.

___

1 Smith et al. (2011) Transcription factors in light and circadian clock signaling networks revealed by genome-wide mapping of direct targets for Neurospora WHITE COLLAR COMPLEX.  Eukaryot. Cell 9:1549–1556.

2 Leleu et al. (2010) Processing and analyzing ChIP-seq data: from short reads to regulatory interactions. Brief Funct Genomics 9: 466-476

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  1. #1 by Scott on June 21, 2011 - 22:22

    How do you purify the nuclei? In your future work, aqueous two-phase extraction may speed things up. Partition of Cell Particles and Macromolecules, by Albertsson PA, 1986.

  2. #2 by Alejandro Montenegro-Montero on June 25, 2011 - 23:24

    We basically do a sucrose gradient…. I’ll look up the reference.

    I’ll take a look at the book. Thanks!

  1. The 2010 Journal Citation Report is in: a molbio view « The MolBio Hut

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