Computational protocol: Profiling DNA supercoiling domains in vivo

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Protocol publication

[…] Microarray text files are read, pre-processed and normalised using the RINGO Bioconductor package in R . Arrays are checked for a uniform hybridisation pattern and the signal intensities are compared across arrays to ensure similarity within fluorophore types (). In rare cases arrays show scratches, drying marks and other artefacts that result in them being discarded from subsequent analyses (a). Furthermore, if the observed signal intensity of the pre-normalised arrays has a non-normal distribution for one/both of the fluorophores then this represents a systematic technical bias and these arrays are discarded.In addition to technical problems that prevent the interpretation of microarray data there are inherent biases that can be corrected for through data normalisation . The difference in signal intensity between Cy3 and Cy5 for a single array (intra-array variation) and between individual fluorophores across arrays (inter-array variation) must be accounted for in order to interpret changes in signal intensity between experiments (b pre-normalisation). Additionally, there is a signal intensity bias that is universal to microarray experiments in which the observed signal intensity ratio (M) varies with the average signal intensity ratio (A) (c pre-normalisation). To correct these biases, normalisation is performed using a variance stabilising algorithm (VSN) from the Limma package (b and c post vsn normalisation). Other normalisation procedures, such as a sequential loess and scale normalisation, give almost identical results in the final analyses (data not shown).To correct for DNA supercoiling independent differences in bTMP binding across array samples, the base-line bTMP bound to genomic DNA is subtracted from bTMP bound in cells. This quantitative measure of bTMP binding allows the comparison of DNA supercoiling across loci in vivo, with positive number showing an enrichment of under-wound DNA and negative numbers showing a depletion of under-wound DNA. All subsequent analysis is performed on the mean of this corrected dataset from duplicate experiments for each experimental condition. […]

Pipeline specifications

Software tools Ringo, limma
Application ChIP-on-chip analysis
Organisms Homo sapiens