7 protocols
normalization data transformation protocol
Data were processed using GeneBASE software. MAT background correction were applied to probe intensities, gene level expression were summarized from the corrected intensities and then quantile normalized. Quantile normalized gene level expression values from 3' RMA ID_REF = VALUE = log2 RMA normalized signal intensity
array scanning protocol
Affymetrix Gene ChIP Scanner 3000 7G
hybridization protocol
Samples were hybridized using Affymetrix hybridization kit materials • Heat cocktails at 99° for 5 minutes, then 42° for 5 minutes centrifuge at max speed for 1 minute (N.B. this deviates from Affy SOP). • Transfer 200μl of hyb solution to each array, then tape holes and parafilm. • Hybridize 16 hours at 45° at 60rpm• Fluidics washing is FS450_0001
labelling protocol
Samples were enzymatically fragmented and biotinylated using the WT Terminal Labeling Kit (Affymetrix)
growth protocol
Mouse brain cells isolated by FACS
sample treatment protocol
Tamoxifen treatment for three weeks to lineage label Shh-responding cells
nucleic acid extraction protocol
Adult mice (6-8 weeks, male) were treated with Tamoxifen (2 x 10mg), to label Shh-responding Gli1+ cells using tdTomato reporter mice. Three weeks later, SVZ, hippocampus and cerebellum were microdissected, dissociated, and FACS isolated for Gli1+ GFAP+ cells. Each sample contains 10-12 mice pooled. Total RNA was isolated from each sample using Trizol and the Qiagen RNeasy Micro Kit Appendix C. Samples were then concentrated using Qiagen RNeasy MinElute Cleanup kit. cDNA synthesis and amplification was performed using WT-Ovation™ Pico RNA Amplification System (NuGEN)