title: GSM7431761: EOMES-ESC-R1; Homo sapiens; OTHER
library_construction_protocol: 10 million hESCs were  fixed with  1% formaldehyde (Thermo Scientific) in PBS without Ca+/Mg+ for 10 min at room temperature . After quenching with  glycine, tubes were centrifuged for 5 min 500g at 4°C. Cells were kept on ice, washed with PBS, centrifuged for 5 min 500g at 4°C, and pellets were frozen in liquid nitrogen and stored at −80°C. Next, cells were vigorously resuspended in 1 ml of ice-cold lysis buffer (10 mM tris (pH 8), 10 mM NaCl, 0.2% NP-40, and 1 tablet of complete protease inhibitor (Roche)], transferred to 9 ml of prechilled lysis buffer, and incubated for 15 min on ice. After centrifugation, cell pellets were resuspended in CutSmart buffer containing 0.5% SDS and incubated for 1 hr at 37°C while shaking at 750 rpm. After, samples were ready for enzymatic digestion. Viewpoint primers were designed either around promoter of EOMES, GATA6, SOX17 and PDX1, depending on the availability of the nearest restriction enzyme sites. Library preparation was then performed using  the Expand Long Range PCR kit (Millipore) under specific PCR conditions (94 °C for 2 min, 16 cycles: 94 °C for 10 s; 59°C for 1 min; 68 °C for 3 min, final extension: 68 °C for 5 min). Amplified DNA was pooled, and primers were removed using AMPure beads (Beckman Coulter,). A second round of PCR was performed using the amplified DNA as a template and overlapping primers to add the P5/P7 sequencing primers and indexes (94 °C for 2 min, 20 cycles: 94 °C for 10 s; 60 °C for 1 min; 68 °C for 3 min and 68 °C for 5 min).