GSM5220242: 4C_SKNMC; Homo sapiens; OTHER
4C was performed to construct the library: 10 million SK-N-MC cells in cell culture were harvested, resuspended in 8.75ml 1X complete medium, and immediately crosslinked in 2% formaldehyde for 10 minutes. Nuclei were isolated from fixed cells and digested by restriction enzyme DpnII (NEB, #R0543) overnight.  Digested DNA was ligated using T4 DNA ligase (NEB, #M2020) and decrosslinked by incubating in proteinase K overnight (Qiagen, #19133). DNA was then purified and selected by weight that is over 30K Dolton, and DNA was digested by the second restriction enzyme Csp6i (Thermo, #ER0211) overnight. DNA was religated using DNA ligase (1U/ul, Invitrogen, #15224017) in low concentration. DNA was further purified and DNA fragments that contain Myc promoter were amplified by PCR reaction with 30 cycles: We chose a transcription start site of Myc gene that is flanked by DPNII and CSP6I cutting site at each end as an anchor (chr8: 127735189-127735615), and we use a pair of primers that contain Truseq indices to amplify regions located to the upstream and downstream of the anchor. The PCR products were further purified, underwent size selection to remove small DNA fragments, and sent for sequencing on Illumina HiSeq 2500 with a depth of 2 million reads. Myc-upstream-SK-N-MC: CAAGCAGAAGACGGCATACGAGAGATGTAGCCGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTTGCCTGCTCTCTGCCAGT. Myc-downstream:  AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTCCACTCTCCCTGGGACTCT