ES cells were grown on ES Medium (DMEM, 15% FBS, Non-essential amino acids, Glutamine, Pen/Strept, 2-mercaptoethanol and LIF) on feeder cells for amplification or on Gelatin for differentiation and experiments. Motor neuron differentiation was done as described (Wichterle & Peljto, Curr Protoc Stem Cell Biol, 2009). Briefly, ES cells were tripsinized and allowed to form aggregates in ANDFK medium (Advanced DMEM/F12, Neurobasal Medium, Knockout-SR, of Pen/Strep, L-Glutamine, and 2-mercaptoethanol. Day 0 is the day of tripsinization. At Day 2, RA and Hh agonist are added.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted with Qiagen RNAeasy at different time points following manufacturer’s instructions.
Label
biotin
Label protocol
RNA was amplified using NuGen Ovation amplification and labeling kit according to manufacturer’s instructions
Hybridization protocol
RNA was hybridized to Affymetrix Mouse Genome 430 2.0 microarrays.
Scan protocol
Arrays were scanned using the GeneChip Scanner 3000.
Description
Gene expression data from ES to Motor Neuron differentiation, Day4 (progenitor motor neuron stage)
Data processing
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 after 8 hours of RA treatment were defined by ranking all probesets by the moderated t-statistic-derived p-value (adjusted for multiple hypothesis testing using Benjamini & Hochberg’s method and setting thresholds of p<0.001 and a fold-change of at least 2.
