(a) Higher-order heterochromatin is disrupted in the genome of SIRT6 KO mice. Whole-genomic DNA was extracted from WT and SIRT6 KO cells, and then digested with the indicated units of micrococcal nuclease (MNase). Digested DNA was separated by gel electrophoresis. This experiment was repeated three times and a representative result is shown. (b) The L1 5′-UTR is hypomethylated in SIRT6 KO cells. Whole-genomic DNA was extracted from SIRT6 KO and WT fibroblasts; methylation levels were quantified using bisulphite primers following bisulphite conversion; n=3, error bars indicate s.d. (c) Multiple heterochromatin formation and maintenance proteins, namely H3K9me3, MeCP2 and KAP1 are specifically depleted from the L1 5′-UTR in SIRT6 KO cells. ChIP was performed using the indicated antibodies in WT and SIRT6 KO cells. Primers spanning the mouse L1 5′-UTR were used for quantification. These experiments were performed three times and representative results are shown. Quantification of the data by qPCR is shown in Supplementary Fig. 5. (d) SIRT6 interacts with multiple heterochromatin associated proteins, including KAP1, MeCP2 and HP1α. SIRT6 was immunoprecipitated from HDF cells and the precipitate was probed with the indicated antibodies. These experiments were repeated three times and representative blots are shown; Input was loaded at 5%. Where appropriate, statistical significance was determined by use of the Student’s t-test.