FUNCTIONAL ANALYSIS OF CTCF DURING MAMMALIAN LIMB DEVELOPMENT_whole transcriptome

Limb buds were dissected from E10.75 mouse embryos and stored in RNAlater reagent (Qiagen), for genotyping. For each replicate, RNA was isolated from pools of 6 limb buds either of wild type or homozygous mutants using RNeasy micro-kit (Qiagen). rRNA was depleted using RiboMinusTM Human/Mouse Transcriptome Isolation Kit (Invitrogen). After cRNA amplification, single or double-stranded cDNA was generated using The GeneChip Whole Transcript Amplified Double-Stranded Target Assay kit (Affymetrix) according to manufacturer's instructions. cDNA was fragmented and labeled using GeneChip WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix) and hybridized to oligonucleotide tiling arrays. The control genomic DNA samples were fragmented with DNAse I. RNA-chip data were computed at the exon level, by averaging the normalized intensities of all probes falling within the exon. As a complement, array data were quantile normalized within cDNA/genomic DNA replicate groups and scaled to medial feature intensity of 10 using TAS software (Affymetrix). For each genomic position, a dataset was generated consisting of all probes mapping within a sliding window of 80 bp. The averaged ratios were plotted along the genomic DNA sequence using Integrated Genome Browser (IGB) software (Affymetrix).