Human PSCs were either grown in primed conditions as above, or switched to naïve media based on N2B27 or mTeSR. N2B27 medium consisted of: 1:1 mixture of DMEM/F12 and Neurobasal medium, with 1% N2 supplement, 2% B27 supplement, 1 mM L-glutamax, 0.1 mM beta-mercaptoethanol, 1x non-essential amino acids and 1x penicillin-streptomycin. N2B27 and mTeSR base media were supplemented with 2iFL: PD0325901 (Stemgent, 0.5 µM), CHIR99021 (Selleckchem, 3 µM), Forskolin (Sigma, 10 µM) and recombinant human LIF (EmdMillipore, 1 ng/ml). LPA (Sigma, 10 µM) was added to naïve media when indicated. The ROCK inhibitor Y-27632 (Stemgent, 5 µM) was used for the first three passages.
Growth protocol
Human primed ESCs and iPSCs were cultured on irradiated MEFs in primed medium (DF12+bFGF) consisting of 80% DMEM/F12, 20% Knockout serum replacement (KSR), 1 mM L-glutamax, 1% non-essential amino acids, 0.1 mM beta-mercaptoethanol, 1% penicillin-streptomycin and 8 ng/ml basic fibroblast growth factor (bFGF) (all from Invitrogen).
Extracted molecule
total RNA
Extraction protocol
total RNA was extracted with RNeasy mini Qiagen kit, and DNAse treated
Label
biotin
Label protocol
100ng was used to generate labelled cDNA using the Illumina specific Ambion TotalPrep kit, stained with Streptavidin-Cy3
Hybridization protocol
biotinylated cRNA was produced from cDNA and hybridized to the Illumina HT-12 v4 gene exression chip O/N at 58oC
Scan protocol
scanned using iScan and expression data extracted using BeadStudio
Description
10002121031_B
Data processing
Data were quantile normalised and log2 transformed using the R/Bioconductor package v.3.1.3 in Rstudio for Mac OSX Normalised values are quantile normalised and log2 transformed intensities
