We use deep sequencing techniques to investigate nucleosome positioning during differentiation, the role of non-coding RNAs in genome organization and the interplay of epigenetic modifications with transcriptional activity.
DNA fragments that are obtained from (1) cDNA synthesis, (2) MNase digestion of chromatin or (3) chromatin immunoprecipitation are cloned into a sequencing library for the Illumina Hiseq platform. These libraries then contain an adaptor for hybridization to the surface of a flowcell and binding sites for the sequencing primer and of course the to be sequenced fragment.
Figure 1: The sequence for each cluster (represented by a single dot) is read via colour-coded nucleotides that added in a stepwise manner. Thus the position and the consecutive fluorophore signals can be used to gain the sequence information. These sequences are mapped to a reference genome and further analyzed depending on the sequencing technique.
After hybridization to the flowcell, the cloned fragments are amplified via bridge- amplification to form dense clusters of identical sequences. Then the sequencing reaction is started with fluorescently labeled nucleotides with a different colour for each base. The nucleotides are stepwise incorporated and the fluorescent signal for each cluster is detected. This is then repeated up to 100 times to gain 100 base pair long sequence reads.
The reads are then mapped to the reference genome and sophisticated analyses, depending on the research topic are applied. For that we use a combination of freely available software tools such as bowtie, cufflinks, tophat, R / biocondutor, galaxy and also commercially available software bundles like genomatix.
Teif VB, Erdel F, Beshnova DA, Vainshtein Y, Mallm JP & Rippe K. (2013).
Taking into account nucleosomes for predicting gene expression.
Methods, DOI: 10.1016/j.ymeth.2013.03.011.
Teif VB, Vainshtein Y, Caudron-Herger M, Mallm JP, Marth C, Höfer T, Rippe K. (2012).
Genome-wide nucleosome positioning during embryonic stem cell development.
Nature Struct. Mol. Biol. 19, 1185-92.
Caudron-Herger M & Rippe K. (2012).
Nuclear architecture by RNA.
Curr. Opin. Genet. Dev. 22, 187-197.
Caudron-Herger M, Müller-Ott K, Mallm JP, Marth C, Schmidt U, Fejes-Tóth K & Rippe K. (2011).
Coding RNAs with a non-coding function: maintenance of open chromatin structure.
Nucleus 2, 410-424.
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