array scanning and feature extraction protocol
Microarrays were imaged on a GCS3000 7G fluorescence scanner (Affymetrix). Feature extraction was performed using Command Console 3.2.3 and hybridization quality assessed with Expression Console 1.1.2 (Affymetrix).
nucleic acid hybridization to array protocol
Affymetrix Exon Array 1.0 ST arrays were hybridized at 45 deg C for 16h, washed and stained with streptavidin-phycoerythrin (SAPE) conjugate on a FS450 automated fluidics station.
nucleic acid labeling protocol
RNA samples were processed for microarray hybridisation according to the GeneChip Whole Transcript Sense Target Labeling Assay (Affymetrix). 2 ug of each sample was depleted of ribosomal RNA using the RiboMinus system (Invitrogen). Double-stranded cDNA was synthesised using random hexamers tagged with a 5' T7 primer, and the products were amplified with T7 RNA polymerase to generate antisense cRNA. Reverse transcription was performed on the cRNA template using SuperScript III to yield single-stranded DNA in the sense orientation, substituting dUTPs for dTTPs, and the cRNA was subsequently degraded via RNase H digestion. cDNA products were then nicked with uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) at sites of first-strand dUTP incorporation, followed by biotin labelling with terminal deoxynucleotidyl transferase (TdT).
iPSCs and human ESCs were cultured in DMEM/F-12 supplemented with: non-essential amino acids, 2mM L-Glutamine, 0.1mM 2-Mercaptoethanol, 20% Knockout Serum Replacement (Life Technologies) and FGF-2 (10ng/ml). Cells were plated on mitotically-inactivated mouse embryonic fibroblasts. Cells were split every 7-14 days (1:3-1:10) or frozen in clumps using dissociation buffer (20% KSR, 1mM CaCl2, 1mg/ml Collagenase IV, 0.25% Trypsin in PBS) or freezing buffer (2M DMSO, 1M Acetamide, 3M propylene glycol in ES cell media).
GNS and NS cells were cultured using serum-free basal media supplemented with B27 and N2 culture supplements (Life Technologies). Growth factors EGF and FGF-2 (20 ng/ml) were added. Culture vessels were coated with Laminin (Sigma) for 3 hr at 10 ug/ml prior to use. Alternatively media was directly supplemented with 1 ug/ml Laminin. GNS cells were routinely grown to confluence, dissociated using Accutase (Sigma), and then split 1:3 to 1:7. Medium was replaced every 3-7 days.
nucleic acid extraction protocol
RNA was extracted with Trizol (Invitrogen) followed by treatment with TURBO DNase (Ambion). RNA quality was assessed on the Agilent 2100 Bioanalyzer.
Cells were dissociated using Accumax (Millipore) and feeder cells depleted.