ChIP-seq Protocol for ATRX and H3.3

For ChIP of ATRX cells were fixed with 2 mM ethylene glycol bis(succinimidyl succinate) (EGS) (Pierce 26103) for 45 min at room temperature in PBS. Formaldehyde was then added to 1% for 20 min and quenched with 125 mM glycine. Chromatin was sonicated to under 500 bp and lysates were immunoprecipitated using 20 μg anti-ATRX antibody (Santa Cruz sc-15408) antibody. DNA was precipitated with 20 μg carrier glycogen. Histone H3.3 chromatin immunoprecipitations were performed using Millipore chromatin Immunoprecipitation Assay Kit (17-295) as per manufacturer's instructions and 10 μg anti-histone H3.3 antibody (Millipore 09-838)45 (link). Telomere binding was assessed by slot blotting and probing with a ³²P-labelled TTAGGG probe. Binding at other loci was assessed by quantitative-PCR (qPCR); primers are shown in Supplementary Table 1. Quantification was performed using ImageJ software and presented as a percentage of the input DNA.