Unknown,Transcriptomics,Genomics,Proteomics

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FUNCTIONAL ANALYSIS OF CTCF DURING MAMMALIAN LIMB DEVELOPMENT_gene expression profiling

ABSTRACT: 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 six limb buds either of wild type or homozygous mutants, using RNeasy micro-kit (Qiagen). cRNA was synthesized according to the manufacturers instructions (Ambion) and hybridized to the GeneChip Mouse Genome 430 2.0 Arrays (Affymetrix), which interrogates ca 39,000 transcripts. Three independent RNA extractions, cDNA synthesis and array hybridizations were performed. The expression arrays data were normalized and scaled to signal intensity of 100 using GCOS 1.2 software (Affymetrix). Expression levels were analyzed using GeneSpring software (Silicon Genetics, Redwood City, CA) and Matlab 2009 (Math Works, Inc., MA). To identify differentially expressed transcripts, pair-wise comparison analysis was performed with GCOS 1.2 software (Affymetrix). A 77% cutoff in consistency of change (at least seven out of nine comparisons were either increased or decreased) was applied. Only genes that satisfied the pair-wise comparison test and displayed ?1.5 fold change in expression were considered for further analysis.

ORGANISM(S): Mus musculus

SUBMITTER: Natalia Soshnikova

PROVIDER: E-MEXP-2977 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Limb buds were dissected from E10.75 mouse embryos, fixed in 1% formaldehyde for 15 min at room temperature, washed 3 times with cold PBS and stored at -800C. Pools of 16 limb buds were used for each ChIP-chip experiment. ChIP was performed according to (Lee et al., 2006) using 2 ?g of anti-CTCF antibodies (A300-543A, Bethyl Laboratories) and EZview Red Protein G/A Affinity Gel (Sigma). Immunoprecipitated and whole cell extract DNA (input) were treated with RNaseA, proteinase K and purified by 2 rounds of extraction with phenol:chloroform: isoamyl alcohol. ChIP and input DNA were amplified using Ligation-Mediated PCR (Lee et al., 2006). PCR was limited to 15 cycles. 1.5 ?g of ChIP and input DNA were fragmented and labeled using GeneChip WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix) and hybridized to the Chromosome 2 and X tiling arrays (Affymetrix). Tiling arrays data were quantile normalized within cDNA/genomic DNA or ChIP/ input replicate groups using R packages, STARR and Ringo (Zacher et al., 2010; Toedling et al., 2007). The ratio of probe intensity between the experiment and the control groups were computed considering the median values over replicates. The ratios were smoothened by computing the running medians with a half window size set to 150 bp and a minimum of 5 probes per window. To identify enriched regions a minimum of 3 consecutive probes with a smoothed ratio exceeding a threshold have been considered. The threshold has been fixed by taking the 99th percentile of the estimated null distribution of the ratios. Only ChIP enriched regions with score log2?1.5 and width ?150 bp were considered for further analysis. As a complement, array data were quantile normalized within ChIP/input replicate groups and scaled to medial feature intensity of 500 using TAS software (Affymetrix). For each genomic position, a dataset was generated consisting of all probes mapping within a sliding window of 250 bp. The averaged ratios were plotted along the genomic DNA sequence using Integrated Genome Browser (IGB) software (Affymetrix).

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FUNCTIONAL ANALYSIS OF CTCF DURING MAMMALIAN LIMB DEVELOPMENT_gene expression profiling

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