title: GSM4828865: TS62_THP-1_S; 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) with co-ordinates from the ABCB1 promoter active in THP-1_R cells (hg38, chr7:87,598,302-87,601,399). 4C sequencing was performed according to the protocol developed by Splinter et al. (6). Briefly, 107 cells were cross-linked with 2% formaldehyde for 10 minutes at room temperature before the reaction was quenched with 0.125M glycine. Cells were lysed with buffer containing 50mM Tris–HCl pH7.5, 150mM NaCl, 5mM EDTA, 0.5% NP-40, 1% TX-100 and 1x complete protease inhibitors (Roche, #11245200). The cross-linked nuclear preparation was then incubated with DpnII. Digestion was confirmed by reversing crosslinking for an aliquot and running on a 0.6% agarose gel. Samples were then ligated overnight at 16°C using T4 DNA ligase (Roche, #799009). Ligation efficiency was again confirmed with 0.6% agarose gel. Crosslinking was reversed and DNA extracted using phenol-chloroform. Samples were then subjected to a second digestion using Csp6I. Ligation was again performed overnight at 16°C using T4 DNA ligase. DNA was then extracted using phenol-chloroform and purified with a QIAquick PCR purification kit (Qiagen, #28104). PCR primers were designed to incorporate 4C primers with a barcode and Illumina adapter sequences: Non-reading primer: CAAGCAGAAGACGGCATACGAGATAGGGTAGGTATTCCACTTTT; Reading primer (THP-1_S): AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCAATGAGATACCAGGTCTGATC; Reading primer (THP-1_R):  AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTAGAGATACCAGGTCTGATC. PCR was performed with Expand Long Template Polymerase (Roche, #11759060001) using 3.2μg of 4C template product and then purified using a High Pure PCR Product Purification Kit (Roche. #11732676001). Library quality was assessed using a 2100 Bioanalyzer (Agilent Technologies). Samples were sequenced with 10% phiX using a MiSeq desktop sequencing system (Illumina) with 75bp single-end settings generating a mean of 1.4M reads per sample.