Illumina MiSeq sequencing; Circularized chromosome conformation capture (4C-seq) in A549 cells after glucocorticoid re-induction
Circularized chromosome conformation capture (4C-seq) in A549 cells after glucocorticoid re-induction
4C-seq was performed with 5 million cells per treatment, cells were grown in DMEM containing 5% fetal bovine serum. A549 cells were grown to approximately 90% confluence according to standard protocols in DMEM medium with 5% FBS. Cells were treated with either 100 nM dexamethasone or 0.1% ethanol. After 4 hours, cells were washed 2x with PBS and cultured in hormone-free medium for 24 hours, after which cells were treated again for 4 hours with either 100 nM dexamethasone or 0.1% ethanol. 4C template preparation was carried out as described in PMID: 22929766. Restriction digests were performed with the enzymes Csp6I (primary, Thermo Fisher Scientific) and DpnII (secondary, Thermo Fisher Scientific). 4C-seq library preparation was performed as described in PMID: 31351925 (except that the DNA was purified with 1.5Ã— AMPure XP beads (Beckman Coulter) after the first PCR). The following primer pairs were used for the inverse PCR: ZBTB16 promoter-proximal VP (fwd: TACACGACGCTCTTCCGATCTAGCAGAGAGGAGTTGAGG, rev: ACTGGAGTTCAGACGTGTGCTCTTCCGATCTTAGTGAACCAGGTGCCAG), ZBTB16 intronic VP (fwd: TACACGACGCTCTTCCGATCTATGTGTGCGTTCATGTATGT, rev: ACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAGGAAAGGTTAGGAAGTGG)