title: GSM2902644: WT_hiPSC_TFAP2A_Prom_1; Homo sapiens; OTHER
library_construction_protocol: 4C-seq assays were performed as previously described (Splinter et al., 2012; Stadhouders et al., 2013; Cruz-Molina et al., 2017) with slight modifications. Approximately 5-10 million hiPSC, hNCC or chicken FNP cells were crosslinked with 1% formaldehyde during 20 minutes and quenched with 0.125M glycine for 10 min. Cells were washed with PBS and resuspended in lysis buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% TX-100 and 1X protease inhibitors) during 10 min on ice.  Following centrifugation for 5 min at 650g (4 °C), nuclei were re-suspended in 0.5 ml of 1.2X restriction buffer with 0.3% SDS and incubated at 37 °C and 900 rpm for 1h. After that, Triton X-100 was added to a final concentration of 2% followed by 1h incubation at 37 °C and 900 rpm. Afterwards, chromatin was digested overnight at 37 °C and 900 rpm with 400 U of NlaIII (R0125L, NEB). NlaIII was inactivated by adding SDS to a final concentration of 1.6% and incubating the mixture for 20 min at 65 °C while shaking (900 rpm). The digested chromatin was transferred to 50-ml tubes and 6.125 ml of 1.15X ligation buffer (50 mM Tris-HCl pH 7.6, 10 mM MgCl2, 1 mM ATP, 1mM DTT) were added. Triton X-100 was also added to a final concentration of 1% and the resulting solution was incubated for 1 h at 37 °C while shaking gently. After that, digested chromatin was ligated with 100 U of T4 DNA ligase (15224-041, Life Technologies) for 8 h at 16 °C, followed by RNAse A treatment (300 μg, Peqlab) for 45 min at 37 °C. Subsequently, chromatin was de-crosslinked with 300 μg of Proteinase K (Peqlab) and overnight incubation at 65 °C. DNA was then purified by phenol/chloroform extraction followed by ethanol precipitation and re-suspension in 100 μl of water. At this step, the digestion and ligation efficiencies were evaluated by analysing a small fraction of the purified DNAs by agarose electrophoresis. The remaining DNA was digested with 50U of DpnII (R0543M, NEB) in 500 μl of 1x NEBuffer DpnII at 37 °C overnight. DNA samples were purified by phenol/chlorophorm extraction, followed by ethanol precipitation. Once purified, DNA samples were re-suspended in 500 μl of water and a second ligation was performed by adding 200 U of T4 DNA Ligase into a final volume of 14 ml 1X ligation buffer followed by overnight incubation at 16 °C. DNA samples were subjected to another round of phenol/chlorophorm extraction and ethanol precipitation, re-suspended in 100 μl of water and purified with a QIAgen PCR purification column (28104, QIAgen). The efficiencies of the second digestion and ligation were also evaluated by agarose electrophoresis. 4C samples were amplified by inverse PCR using primers located within different human (TFAP2A promoter, ENh480, Enh100) or chicken (3' TFAP2A) genomic regions and that included illumina adaptor sequences and short barcodes. The inverse PCRs were performed with the expand long template PCR system (11681842001, Roche) and 30 amplification cycles (94°C 2 min, 30x [94°C 10 sec, 60°C 1 min, 68°C 3 min], 68°C 5 min). The inverse PCR primers were designed as previously described (Stadhouders et al., 2013). Once amplified, 4C-seq libraries were sequenced on the HiSeq2500, generating reads of either 74 or 100 bp in length.
identifiers.namespace: GEO
identifiers.id: GSM2902644