Tn 16

Cells were fixed with 80% acetone at −20°C for 10 min or 2.5% formaldehyde (Wako; 063-04815) at room temperature for 10 min. formaldehyde-fixed cells were permeabilized with 0.5% Triton X-100 in PBS. Fixed cells were blocked in 2% BSA in PBS for 30 min. Cells were incubated at 37°C for 1 h with each of the primary and secondary antibodies. For chromosome spreads, HT1080 tetR–EYFP–CENP-B N21 cell line (Fig. 8C) was treated with 350 nM TN-16 (Wako) (Kitagawa et al., 1995 (link)) for 3 h in the culture medium. Mitotic cells were harvested, incubated in 0.075 M KCl for 10 min on ice, and then spread on a cover-glass using a Cytospin3 centrifuge (Shandon). The subsequent immunostaining and fluorescence in situ hybridization (FISH) were performed to confirm HAC formation according to a previously reported method (Ikeno et al., 1998 (link); Ohzeki et al., 2002 (link)). For Halo-tag labeling, cells were cultured in the presence of 10 nM Halo-TMR-Ligand (Promega; G8251) or 10 nM Halo-Biotin-Ligand (Promega; G8281).

Preparation of chromosome spreads and subsequent fluorescence in situ hybridization (FISH) was described previously [46 (link)], except that we enriched for mitotic cells by treatment with 300 nM TN-16 (FUJIFILM Wako Pure Chemical Corp., Doshomachi Osaka, Japan) for 2 h. An evaluation of HAC formation was performed, as described previously [33 (link)].
The ChIP and subsequent qPCR and competitive PCR were described previously [45 (link)]. Anti-HP1α was purchased from abcam (Cambridge, UK; ab77256). PCR primers were from the literature [40 (link)], except N11F5 (5′-GGGATCACTAGCAATAAAAGGTAGAC-3′) and N11R6 (5′-TCCTTCTGTCTCGTTTTTATGGC-3′) used for competitive PCR of tetO vs. lacO, and N11F8 (5′-AGACAGAAGCATGCTCAGAAAC-3′) and N11R11 (5′-CTACCTTTTATTGCTAGTGATCCC-3′) for CENP-B box wild-type vs. mutant.