ESCs and EBs were cultured and differentiated as described previously (Wiles et al., Exp Cell Res 1999;247:241-8). Neural induction using SB431542 (Cambridge Bioscience) was adapted from earlier protocols (Ying et al., Nat. Biotechnol. 2003; 21:183-6; Chambers et al., Nat Biotechnol 2009;27:275-80). Briefly, ESCs were plated on fibronectin-coated coverslips or gelatin-coated 6-well clusters at a density to achieve 80-90% confluence after a further 24h, after which growth medium was d with N2B27 medium containing 5uM SB431542. RG-NSCs were generated according to Conti et al. (PloS Biol 2005;3:e283) and differentiated using either a tri-potential differentiation protocol (Glaser et al., PloS One 2007;2:e298) or for astrocyte differentiation, in NS-A/Euroclone medium (Cadama) supplemented with 10% FCS or 20ng/ml BMP4.
Extracted molecule
total RNA
Extraction protocol
Total RNA isolation, reverse transcription and PCR analyses were carried out as previously described (Johnson et al., PLoS Biol, 2008;6:e256).
Label
biotin
Label protocol
RNA was labelled using a TotalPrep RNA Amplification Kit (Ambion).
Hybridization protocol
Standard Illumina hybridization protocol to Illumina Mouse Ref-8 v2 BeadArrays.
Scan protocol
Standard Illumina scanning protocol.
Description
4613963049_F REST KO replicate 3
E14Tg2a line: See Hooper et al., 1987, Nature, 326, 292-295 (PMID 3821905).
Data processing
The data were normalized using quantile normalization using the Bioconductor BeadArray library in R. Differential expression was calculated using the limma library.
