Total RNA was isolated from 0-day (undifferentiated cells) and 7- and 14-day EBs (differentiated cells) using RNeasy kits (Qiagen, Hilden, Germany, http://www1.qiagen.com). Briefly, cells were lysed in 600 μl/well (six-well plate) RLT buffer containing 10 μl/ml β-ME and subsequently homogenized by filtration through a QIAshredder. One volume of 70% ethanol was then added to the homogenate, and the sample was applied to an RNeasy mini column. On-column DNase digestion was carried out during the isolation using an RNase-Free DNase Set (Qiagen). After a series of washes, RNA was eluted in RNase-free water. First-strand cDNA synthesis was subsequently carried out using approximately 1 μg of total RNA and Superscript II reverse transcription, following the manufacturer's instructions (Invitrogen). Polymerase chain reaction (PCR) mixtures were prepared as described (Invitrogen); final MgCl2, dTNP, and oligonucleotide concentrations used were 1.5 mM, 200 μM, and 200 nM, respectively. Amplification parameters were as follows: 1 cycle at 94°C for 2 minutes; 35 cycles at 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute; and 1 cycle at 72°C for 10 minutes. Quantitative real-time PCR was performed using a Stratagene Mx4000 and a Brilliant Cyber Green qPCR Core Reagent Kit (Stratagene, La Jolla, CA, http://www.stratagene.com) following the manufacturer's instructions. Briefly, 1.5 mM MgCl2, 800 μM dNTPs, 200 nM oligonucleotide primers, 8% glycerol, 5% dimethylsulfoxide, 20 nM reference dye, and × 0.5 SYBR green dye were used in reaction mixtures. Samples were normalized against GAPDH. Primer sequences were as follows: XIST 5′, agctcctcggacagctgtaa; XIST 3′, ctccagatagctggcaacc; OCT3/4 5′, cttgctgcagaagtgggtggaggaa; OCT3/4 3′, ctgcagtgtgggtttcgggca; CRIPTO 5′, acagaacctgctgcctgaat; CRIPTO 3′, atcacagccgggtagaaatg; REX-1 5′, tgaaagcccacatcctaacg; REX-1 3′, caagctatcctcctgctttgg; NANOG 5′, caaaggcaaacaacccactt; NANOG 3′, tctgctggaggctgaggtat; STAT3 5′, tttcacttgggtggagaagg; STAT3 3′, ggctacctgggtcagcttcag; UTF-1 5′, accagctgctgaccttgaac; UTF-1 3′, ttgaacgtacccaagaacga; GAPDH 5′, gagtcaacggatttggtcgt; GAPDH 3′, ttgattttggagggatctcg.