tissue: whole back muscle genotype/variation: SIX1/4 mutant
Treatment protocol
no treatment
Growth protocol
No growth protocol, the tissue is extracted directly from E15 or E18 embryos
Extracted molecule
total RNA
Extraction protocol
RNA extraction from Pax7-nGFP+ FACS-sorted cells was performed using the Qiagen RNeasy microkit directly after isolation. Whole back muscles required a tissue lyser lyse step in Trizol solution.
Label
biotin
Label protocol
50 ng (E18.5 whole back muscles), 2 ng (E15.5 FACS-sorted cells and whole back muscles) or 0.21 ng (E18.5 FACS-sorted cells) of total RNA were reverse-transcribed following the Ovation PicoSL or PicoV2 WTA System (Nugen). Briefly, the resulting double-strand cDNA was used for amplification based on SPIA technology. After purification according to Nugen protocol, 5 μg of single strand DNA was used for generation of Sens Target DNA using Ovation Exon Module kit (Nugen). 2.5 μg of Sens Target DNA were fragmented and labelled with biotin using Encore Biotin Module kit (Nugen).
Hybridization protocol
The cDNA was then hybridized to GeneChip® Mouse Gene 1.0 or 2.0 ST (Affymetrix) at 45 °C for 17 h. After overnight hybridization, the ChIPs were washed using the fluidic station FS450 following specific protocols (Affymetrix).
Scan protocol
the ChIPs were scanned using the GCS3000 7G. The scanned images were then analyzed with Expression Console software (Affymetrix) to obtain raw data (cel files) and metrics for quality controls
Data processing
Normalisation with R, by project Project 1 : WT1_Muscle_E15,WT2_Muscle_E15,WT3_Muscle_E15,Mut1_Muscle_E15,Mut2_Muscle_E15,Mut3_Muscle_E15 → mogene20st_Mm_ENTREZG from Brainarray, version 20, Matrix 3 WT1_Muscle_E18,WT2_Muscle_E18,Mut1_Muscle_E18,Mut2_Muscle_E18 → mogene10st_Mm_ENTREZG from Brainarray, version 20, Matrix 2 Then we conserved only common genes between the two lists. Project 2 : WT1_PAX7_E15, WT2_PAX7_E15, WT3_PAX7_E15, Mut1_PAX7_E15, Mut2_PAX7_E15, Mut3_PAX7_E15, WT6_PAX7_E18, WT7_PAX7_E18, WT8_PAX7_E18, Mut6_PAX7_E18,Mut7_PAX7_E18,Mut8_PAX7_E18 mogene20st_Mm_ENTREZG from Brainarray, version 19, Matrix 1 We strictly follow the Brainrray recomandations for the normalisation by using the affy package they provide for each version of their custom CDF files.
