Atto647n nhs ester

In order to prepare Atto-647N-labeled LFY for in vitro microscopy experiments, LFY at a concentration of 1 μM was labeled with ATTO 647N NHS ester (AD 647N-31, ATTO-TEC) according to the manufacturer’s instructions. Free ATTO 647N dye was removed using a Zeba MicroSpin desalting column 7K MWCO (89877, Thermofisher). LFY-ATTO 647N was spiked into unlabeled LFY at a 1:100 ratio, and phase separation was induced by mixing with 2% PEG 20,000 (813000, Fluka) at a salt concentration of 100 mM NaCl. After 15 minutes of incubation at 4°C, the samples were pipetted on a microscopy slide, which was precoated with PEG 8000. All experiments were carried out at room temperature within 20 min after pipetting.

About 12-mer repeats of the 601 nucleosome positioning sequence (26 (link)), separated by either 30 bp linker (a177) or 50 bp linker (a197), flanked by EcoRV sites and a unique BsaI restriction site at the 3′ end were cloned into plasmids and purified from bacterial culture by size exclusion chromatography followed by a phenol/chloroform extraction. The plasmids were then subjected to a restriction digest and the 12 × 601 sequences were purified by polyethylene glycol (PEG) precipitation. For immobilization and visualization, an oligonucleotide (5′-ph-CAGCTAGTCTGCT-(amine-linker)-CAGATATCGTCG-3′-Biotin) was labeled using Atto647N-NHS ester according to the manufacturer's (AttoTec) protocol. The labeled oligonucleotide was then annealed to its complementary DNA strand (5′-CGACGATATCTGAGCAGACTA-3′) and ligated to the 12 × 601 array DNA with T4 DNA ligase for 1 h at room temperature. The excess dsDNA was removed by purification with QIAquick spin columns (Qiagen), and the final 12 × 601 DNA was finally purified by ethanol precipitation (Supplementary Figure S1).