7 protocols
Microarrays were imaged on a BeadArray Reader fluorescence scanner (Illumina). BeadStudio software (Illumina) was used to infer methylation scores from image intensities.
Bisulfite-converted DNA was hybridized to the Illumina Infinium450K Human Methylation Beadchip.
1ug of DNA was bisulfite converted using the EZ DNA methylation kit (Zymo Research). Labeled nucleotides are incorporated by single-base extension, where methylated (cytosine) bases are labeled with Cy3 and unmethylated (thymine) bases with Cy5.
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.
Cells were dissociated using Accumax (Millipore) and feeder cells depleted.
Genomic DNA was extracted using the DNeasy kit (Qiagen).
iPS cells 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).