Chip grade a g agarose beads

Primary human T cells (10⁷) were activated overnight by anti-CD3 plus anti-CD28 (both at 0.5 μg/ml) in the presence or absence of Cy3-TelC-labelled APC-derived enriched supernatants containing telomeres derived from ionomycin activated TelCy3 live-labelled APCs. Cells were cross-linked 10 min with 1% formaldehyde, lysed and their nuclei digested with MNase-based Thermofisher Pierce Agarose Chip kit (Pierce™ Chromatin Prep Module, 26158). Digested chromatin was immunoprecipitated with polyclonal anti-POT1 or control rabbit IgG antibodies at 4C on a rotary shaker overnight followed by incubation with Chip-grade A/G agarose beads (sc-500775; Santa Cruz Biotechnology) at 4C for 3 hours. Presence of APC-derived telomeres in control and POT-1 (1:100; PA-66996, ThermoFisher) immunoprecipitated T-cell reactions was quantified as Cy3 absorbance emission of triplicate wells using a micro-plate reader. Presence of APC-derived telomeres was further confirmed by adding DNAse directly to the POT1 IPs for 10 min prior to reading. Background fluorescence was calculated in parallel POT1 immunoprecipitation reactions from activated CD3⁺ CD4⁺ CD27⁺ CD28⁺ T cells not treated with APC-derived telomeres.