Computational protocol: Saltatory remodeling of Hox chromatin in response to rostro-caudal patterning signals

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Protocol publication

[…] Total RNA was extracted from ES cells or embryoid bodies using Qiagen RNAeasy kit. For quantitative PCR (qPCR) analysis, cDNA was synthesized using SuperScript III (Invitrogen) and amplified using SYBR green brilliant PCR amplification kit (Stratagene) and Mx3000 thermocycler (Stratagene). For GeneChip expression analysis, RNA was amplified using Ovation amplification and labeling kit (NuGen) and hybridized to Affymetrix Mouse Genome 430 2.0 microarrays. Expression experiments were performed in biological triplicate for each analyzed time-point. Arrays were scanned using the GeneChip Scanner 3000. Data analysis was carried out using the affylmGUI BioConductor package . GCRMA normalization was performed across all arrays, followed by linear model fitting using Limma . Differentially expressed genes were defined by ranking all probesets by the moderated t-statistic-derived p-value (adjusted for multiple testing using Benjamini & Hochberg’s method) and setting thresholds of p<0.001 and a fold-change of at least 2. All arrays were submitted to the NIH GEO database under accession numbers GSE19372 (RA/Hh-dependent motor neuron differentiation timeseries) and GSE39422 (response to Cdx2 expression in motor neuron progenitors). […]

Pipeline specifications

Software tools affylmGUI, GC–RMA, limma
Application Gene expression microarray analysis
Organisms Mus musculus
Chemicals Tretinoin