7 protocols
Scanning was carried out according to manufacturer specifications.
Labeled genomic DNA fragments were hybridized to Affymetrix Genome-Wide Human SNP 6.0 arrays.
500 ng of genomic DNA was subjected ty restriction enzyme digestion by NspI and StyI and ligated to adapters. Ligation products were PCR amplified with a biotinylated primer, fragmented and labeled with Cy3 for single-channel hybridization.
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).
Cells were dissociated using Accumax (Millipore) and feeder cells depleted.
Genomic DNA was extracted using the DNA Wizard kit (Promega).
GNS cells were cultured using serum-free basal medium supplemented with B27 and N2 (Life Technologies) and growth factors EGF and FGF-2 (20 ng/ml). Culture vessels were coated with Laminin (Sigma) for 3 h 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.