7 protocols
AccessionNameType
P-MTAB-28190
scanning
Microarrays were imaged on a BeadArray Reader fluorescence scanner (Illumina). BeadStudio software (Illumina) was used to infer methylation scores from image intensities.
P-MTAB-28189
hybridization
Bisulfite-converted DNA was hybridized to the Illumina Infinium450K Human Methylation Beadchip.
P-MTAB-28188
Labeling
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.
P-MTAB-28184
grow
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.
P-MTAB-28186
specified_biomaterial_action
Cells were dissociated using Accumax (Millipore) and feeder cells depleted.
P-MTAB-28187
nucleic_acid_extraction
Genomic DNA was extracted using the DNeasy kit (Qiagen).
P-MTAB-28185
grow
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).