Telomere-Binding Interactome Identification

Telomere pull-downs were done as previously described (43 (link)). In brief, chemically synthesized oligonucleotides with either TTAGGG or TGTGAG repeats were annealed with their complementary oligonucleotides. The dsDNA was phosphorylated with 100 units PNK (Thermo) for 2 h at 37°C and ligated overnight with 20 units T4 ligase (Thermo) at room temperature (RT). The mixture was cleaned by chloroform phenol extraction and the purified DNA was incubated with biotin-dATP (Jena Biosciences) and 60 units Klenow fragment (Thermo) at 37°C overnight. The DNA was re-buffered using a G50 Spin column (GE Healthcare) according to the manufacturer’s instructions. Around 25 μg biotinylated DNA was immobilized on Streptavidin Dynabeads MyOne C1 (Thermo) and incubated with trypanosome PCF whole cell lysate obtained by lysis in modified RIPA buffer [50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Igepal CA-630, 0.1% Sodium Deoxycholate, Protease inhibitor cocktail (Roche)]. The binding reaction was performed in protein binding buffer (PBB) (150 mM NaCl, 50 mM Tris–HCl pH 7.5, 5 mM MgCl₂, 0.5% Igepal CA-630) in the presence of 10 μg sheared salmon sperm DNA (Ambion) at 4°C for 2 h under slight agitation. Unbound proteins were washed with PBB three times and the bound fraction eluted with 1× lithium dodecyl sulfate (LDS) buffer (Thermo).