3C assays were performed as referred in27. Mouse brain tissue was processed to get single cells samples. 10 millions isolated cells were treated with lysis buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.3% IGEPAL CA-630 (Sigma-Aldrich, I8896), 1X protease inhibitor cocktail (cOmplete, Roche, 11697498001)) and nuclei were digested with HindIII endonuclease (Roche, 10798983001). Subsequently, DNA was ligated with T4 DNA ligase (Promega, M1804). A set of locus specific primers (see table) was designed with the online program primer3 v. 0.4.028, each one close to a HindIII restriction site. These primers were used to make real time quantitative PCRs using a BioRad CFX96 Real Time System with SsoFast EvaGreen Supermix (BioRad 172-5203), in order to measure the relative enrichment in each ligation product. The primer designed within the LD-block was used as fixed primer. The quality of all primer pairs was measured using serial dilutions of a BAC DNA that encompass the regions of interest (RP23-268O10, RP23-96F3). PCR values were normalized by means of control primers designed in the Ercc3 gene locus. The average of four independent experiments was graphically represented.
