7 protocols
AccessionNameType
P-MTAB-28176
scanning
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).
P-MTAB-28175
hybridization
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.
P-MTAB-28174
labeling
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).
P-MTAB-28171
grow
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).
P-MTAB-28170
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-28173
nucleic_acid_extraction
RNA was extracted with Trizol (Invitrogen) followed by treatment with TURBO DNase (Ambion). RNA quality was assessed on the Agilent 2100 Bioanalyzer.
P-MTAB-28172
specified_biomaterial_action
Cells were dissociated using Accumax (Millipore) and feeder cells depleted.