Circular chromatin conformation capture (4C-seq)

4C-seq was performed as previously described⁸³ with slight modifications. 4C-seq data were replicated for each condition, using male tissues in a first replicate and female tissues in the second replicate. Briefly, 5 million nuclei per biological condition were purified using same steps described in the FANS approach^56,57, except that percentage of formaldehyde was increased up to 2%. Purified nuclei were digested O/N with the first restriction enzyme (DpnII, New England Biolabs) and posteriorly subjected to an overnight ligation with T4 DNA Ligase (New England Biolabs). Subsequently, chromatin was de-crosslinked and purified after proteinase K and RNAse A treatment. A second digestion using Csp6I (Thermo Scientific) was performed O/N, followed by final O/N ligation with T4 DNA Ligase and DNA purification using phenol/chloroform extraction after sample volume reduction by using 90% under-saturated phenol (UPT phenol). The resultant 4C DNA template was used to generate 4C-seq libraries by performing a PCR (Long Template PCR system, Roche) with target-specific designed reading and non-reading primers (see table below) containing Illumina sequencer adapters. For primer design, a region surrounding the TSS of the gene of interest (±2 kb) was retrieved and primers were designed using SnapGene (v.1.1.3) for regions that fulfilled the following criteria: distance between DpnII restriction site and the consecutive Csp6I restriction site >350 bp; distance between DpnII restriction site and the following DpnII restriction site after Csp6I >500 bp and <1500 bp (Supplementary Table ¹). A primer validation step was included to verify their specificity. Then, generated 4C-seq libraries were purified with SPRI select beads (Beckman) to discard primer dimer DNA products and 4C-seq DNA template were quantified using Bioanalyzer and pooled equimolarly for sequencing using 50 bp single-end Hiseq 4000 sequencer (IGBMC Genomeast platform).