Computational protocol: DNA methylation directs genomic localization of Mbd2 and Mbd3 in embryonic stem cells

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Protocol publication

[…] Microarray analysis was performed as previously described (). Total RNA from control (EGFP KD), Dnmt1 KD, Tet1 KD, Mbd3 KD, Mbd2 KD, or Chd4 KD ES cells was subjected to RNA amplification and labeling using the Low Input Quick Amp Labeling Kit (Agilent, Santa Clara, CA, USA). Following this procedure, 50 picomoles of cRNA was used for fragmentation and hybridization on Agilent 4 × 44K mouse whole-genome microarrays. Slides were scanned on a DNA microarray scanner (Agilent G2565CA), and fluorescence data were obtained using Agilent Feature Extraction software. The expression data from biological replicates were fit to a linear model using the Bioconductor package limma (; ; , RRID:SCR_010943), and analyzed as previously described (). Datasets were visualized through Java TreeView (, RRID:SCR_013503). Tip60 KD and Brg1 KD microarray data was downloaded from GSE31008 () and compared to averaged microarray data for fold change of Dnmt1 KD, Tet1 KD, Mbd3 KD, Mbd2 KD, and Chd4 KD. Correlation coefficients for all genes in pairwise factor KD were calculated in R. […]

Pipeline specifications

Software tools Agilent Feature Extraction, limma, TreeView
Application Gene expression microarray analysis
Organisms Mus musculus
Chemicals Cytosine