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ChIP assays were performed following the manufacturer's protocol (Upstate Biotechnology, Lake Placid, NY) using 2 × 106 MCF-7 or MDA-MB-231 cells. An anti-c-Myc antibody (sc-764, Santa Cruz Biotechnology, Inc) or an anti-CTCF antibody (sc-15914, Santa Cruz Biotechnology, Inc) was used to precipitate DNA-protein complexes, and the normal isotype-matched IgG from the same species was used as negative control. The PCR primers used for detecting DNA fragments containing E boxes are: E box X: ChIPX-FW: 5'-AATGCAAAGA CCGTCCGCTG CCA-3'and ChIPX-RV: 5'-TCCACCCCTC AGCCCCAGTG TTT-3'; E box Y: ChIPY-FW: 5'-TGAAGGGCTC CTCCAGCACG-3' and ChIPY-RV: 5'-TGAGGGACCG AGTGGGCGAA-3', and non E box: ChIPNE-FW: 5'-CGAGAGACGC TTGGCTCTTT CTGT-3' and ChIPNE-RV: 5'-GCCCAGTTAT CTGAGAAACC CCAC-3'. The amplified PCR product size was 233 bps for the detection of E box X, 146 bps for E box Y, and 214 bps for non E box, respectively. The band densities of the targeted PCR products were quantified with the software QUANTITY ONE version 4.5.1 (Bio-Rad, Hercules, CA).

The PCR amplification program was optimized for single-stranded DNA and included an initial denaturation at 95°C for 10 min, followed by denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 30 s for 35 cycles. Amplified products were separated in a 1.5% agarose gel electrophoresis, and the band densities were quantified with the software QUANTITY ONE version 4.5.1 (Bio-Rad).

It's not the most intuitive editing software I've ever used, with a few odd quirks, but it is powerful. If you are comfortable with editing photos and creating a design that you like, this software will be perfect for you.

All software and documentation are Copyright 2018 Cisco Systems, Inc. and/or its affiliates. All rights reserved.Content except where otherwise noted is Copyright 2018 Cisco Systems, Inc. and/or its affiliates.

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One of the drawbacks to this approach was that we needed to recompile every time we made even a small change to the code. This slowed everything else we were doing down and because we couldn't always have someone waiting for the application to finish building. 827ec27edc