7 protocols
Title: Affymetrix CEL analysis. Description:
hybridization protocol
Affymetrix Generic Hybridization
nucleic acid labeling protocol
Labeling of samples was carried out according to the manufacturer’s standard protocol
normalization data transformation protocol
All CEL files in the experiment were preprocessed using the Robust Multichip Average (RMA) *alogrithm in the R "affy" package. * Irizarry et al. (2003) Summaries of Affymetrix GeneChip probe level data. Nulceic Acids Res 31, e15.
Embryos at E10.5 were obtained from timed matings of Smchd1 heterozygote intercrosses and detection of a vaginal plug was counted as embryonic day (E) 0.5. Embryos were dissected and then sexed and genotyped by PCR from embryo fragments.
Total RNA was isolated from individual E10.5 embryos using RNeasy kit (Qiagen) according to the manufacturer's instructions. Total RNA was eluted with 2x30ul RNase-free water. Concentration of total RNA was measured with NanoDrop spectrophotometer. Quality and integrity of total RNA was checked on Bioanalyser (Agilent Technologies). Good quality total RNA was divided into 10µg aliquots, snap-frozen on dry ice, and stored at -80 degrees C until use.
The Smchd1 mutant allele used in this study is the MommeD1 null allele. The MommeD1 (for Modifier of Murine Metastable Epiallele) mutation was identified in an ENU mutagenesis screen for modifiers of epigenetic reprogramming as a semi-dominant suppressor of variegated expression from a GFP transgene. Mice carrying the Smchd1 mutant allele were maintained as heterozygotes in the FVB/N background, where the allele was originally isolated.