NextSeq 500 paired end sequencing; Circularized chromosome conformation capture (4C-seq) in A549 and U2OS cells stably expressing rat GRα
Circularized chromosome conformation capture (4C-seq) in A549 and U2OS cells stably expressing rat GRα
Cells were cultured to confluency and treated for 90 min with 1μM dexamethasone. 4C experiments were performed as previously described (van de Werken et al., Methods Enzymol, 2012. 513: p. 89-112.), using 5x10^6 cells. 4-bp cutters were used as primary (CviQI, Thermo Fisher Scientific) and secondary (DpnII, Thermo Fisher Scientific) restriction enzymes.  For each cell line 4C libraries were generated in two biological replicates.  Based on 4C-libraries we amplified a total of 1.2 μg of DNA by inverse-PCR using viewpoint-specific primers (fwd: CTACACGACGCTCTTCCGATCTAATGTTCAGGTGTGGGAGTAC, rev: CAGACGTGTGCTCTTCCGATCTGCCCTCTGCCTCTTGTTAGG), which include adaptors for subsequent high throughput sequencing (Illumina HiSeq2500).
Experimental Factor: A549
cell line