Southern blot on genomic DNA was performed as described in Thijssen et al.63 (link). For major satellite analysis 200 ng of genomic DNA were digested with HpyCH4IV (New England Biolabs) for 1h at 37 °C, while for minor satellites 500ng of gDNA were digested with HpaII (New England Biolabs) and 300 ng with MspI (New England Biolabs), both O/N at 37 °C. Digested samples were separated for 5 h on 1% agarose gel. Gels were then denaturated in a 1.5 M NaCl and 0.5 M NaOH solution for 20 min and neutralized with 0.5 M Tris-HCl pH 7.5 and 1.5 M NaCl for 40 min. Transfer was performed O/N on Hybond-N+ membranes (GE Healthcare) in SSC 20X. After ultraviolet crosslinking, membranes were pre-hybridized in SSC 6X, Denhardt 5X and 0.1% SDS for 1 h at 42 °C and hybridized with 32P-labelled probes for 2 h at 42 °C. After membrane washing, signals were detected using FLA 7000 phosphorimager (Fuji). Images were then analyzed with ImageJ (imagej.nih.gov/ij) to perform linescan for major satellites and intensity ratio HpaII/MspI for the lower six bands of each lane for minor satellites. Probe used: Major satellites 5′-CAC GTC CTA CAG TGG ACA TTT CTA AAT TTT CCA CCT TTT TCA GTT-3′ and minor satellites 5′-ACA TTC GTT GGA AAC GGG ATT TGT AGA ACA GTG TAT ATC AAT GAG TTA CAA TGA GAA ACA T-3′.
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