GSM1200407: HMEC; Homo sapiens; OTHER
4C was performed as in Gheldof et al. with minor modifications. HMEC, MCF7 and MDA-MB-231 cells (2 x 107) were fixed in 2% formaldehyde in fresh medium for 10 min at room temperature, followed by quenching with 0.125 M glycine. Fixed cells were scraped from culture plates, spun, (750 x g for 10 min), and the frozen pellets were stored at -80°C until lysis. Cells were resuspended in ice-cold lysis buffer (0.2% IGEPAL CA-630, 10 mM NaCl, 10 mM Tris HCl) with SigmaFast complete protease inhibitor tablet (Sigma-Aldrich, St. Louis, MO) and lysed for 30 min on ice. After recovery of nuclei by centrifugation (2000 x g for 5 minutes), nuclei were washed twice in cold 1.2X NEB buffer 2 and resuspended in the same buffer. Nuclei were incubated in the presence of 0.3% SDS for 1h at 37°C with shaking at 950 rpm, followed by the addition of Triton X-100 to 1.8% for 1h at 37°C with shaking at 950 rpm. Nuclei were digested with 1500 U of HindIII (New England Biolabs Ipswich, MA) overnight at 37°C with shaking at 950 rpm. 200 μl of digested nuclei were removed for assessing digestion efficiency by qPCR. The restriction enzyme was inactivated by the addition of 1.6% SDS and was incubated at 65°C for 20 min. The digested nuclei were diluted in 7ml of 1.1X T4 DNA ligase buffer in the presence of 1% Triton X-100 and incubated for 1h at 37°C. Ligation was performed by adding 800 U of T4 DNA Ligase (2,000,000 U/ml; New England Biolabs) to the diluted mixture of digested nuclei and incubating in a 16°C H2O bath for 4 hours followed by a 30 min incubation at room temperature. To reverse cross-links, proteinase K was added to a final concentration of 100 μg/ml and incubated overnight at 65°C. Samples were incubated with 0.5 μg/ml of RNase A at 37°C for 1h and purified by phenol-chloroform extraction followed by ethanol precipitation. DNA concentration was measured using a Qubit® 2.0 Fluorometer (Life Technologies). A total of 16 inverse PCR reactions with 200 ng input per 4C template were performed for each library with primers that included Illumina adapter sequences and custom barcodes. All PCR reactions were performed with Expand Long Template PCR system (Roche, Indianapolis, IN). Excess primers were removed by gel extraction. HMEC, MCF7 and MDA-MB-231 4C libraries were analyzed on a MultiNA microchip electrophoresis system (Shimadzu Columbia, MD) and mixed in equimolar amounts. Multiplex sequencing was performed on an Illumina genome analyzer IIx (Illumina, San Diego, CA) using variable length barcodes (TC and AGT for HMEC, CAGT for MCF7, GTCCT for MDA-MB-231).