title: GSM5292623: TS103_352T1B_D_FOXC1_high_V4_TGACCA_CAAGCTGACTATATAGTAC; Homo sapiens; OTHER
library_construction_protocol: 4C primer sequences and enzyme combinations were selected using the University of Chicago online tool: (http://mnlab.uchicago.edu/4Cpd). The viewpoint VP4 placed at the FOXC1 promoter was  chosen for studying the near bait, cis and trans interactions between the locus and the genome. 4C templates were prepared as described previously (Splinter et al., 2012). In brief, 1 x 107 AML cells were cross-linked with 2% formaldehyde for 10 min at room temperature before the reaction was quenched with 0.125M glycine. Cells were lysed in 4C-lysis buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 5mM EDTA, 0.5% NP-40, 1% TX-100 and 1x complete protease inhibitors, Roche) and nuclei were isolated. Cross-linked DNA was digested with the primary restriction enzyme Csp6I (Thermo Scientific) recognizing a 4-bp restriction site. This was followed by proximity ligation overnight at 16°C by the T4 DNA ligase (Sigma-Aldrich) after which cross-links were removed. DNA was extracted using phenol- chloroform and samples were then subjected to a second digestion with the 4-bp secondary restriction enzyme DpnII (New England Biolabs), followed by a second proximity ligation overnight at 16°C. DNA was then extracted using phenol-chloroform and purified using the QIAquick PCR purification kit (QIAGEN). PCR primers were PAGE purified from IDT and contained the P5/P7 Illumina adapter sequence, a barcode for sample identification and the specific viewpoint restriction enzyme sequence. PCR was performed with the Expand Long Template polymerase (Roche) using 3.2μg of 4C template product, and then purified using three columns per sample of the High Pure PCR Product Purification Kit (Roche) that separates the PCR products from the adaptor-containing primers. Thermocycling conditions were: 94°C for 2min, 30 cycles of (94°C for 10s, 55°C for 1min, 68°C for 3min), 68°C for 5min. Library quality was assessed using a 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA). Samples were sequenced with 10% phiX using a MiSeq desktop sequencing system (Illumina, San Diego, CA) with 75-bp, single-end settings generating around 1 million reads per sample.