Proteogenomics analysis reveals specific genomic orientations at distal regulatory regions composed by non-canonical histone variants

Histone variants (H3.1, H3.3, H2A, and macroH2A) were studied about their proteomic and genomic distribution in Hela cells Overall design: HeLa S3 cells stably expressing either FLAG-tagged H3.1, and H3.3 were grown in suspension in Joklik media containing 10% newborn calf serum (Hyclone), 1% GlutaMAX (Invitrogen), and 1% penicillin-streptomycin, and cells were harvested at log phase. Nuclei were isolated, mononucleosomes were subsequently obtained from these cell lines. Cells were lysed in hypotonic TMSD buffer to isolate nuclei, which were then digested with micrococcal nuclease. The resulting mononucleosomes were immunoprecipitated with anti-FLAG M2-agarose beads (Sigma) and eluted with FLAG peptide (Sigma)