Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Approximately 2 mg of nuclear extracts was incubated with primary antibody overnight at 4°C. Extract and antibody were added to protein A and protein G Dynabeads™ (ThermoFisher Scientific, #10002D and 10004D) for 4 h at 4°C, washed with PBST, and eluted with 0.1 M glycine (pH 2.3). Eluates were neutralized with 1.5 M Tris buffer (pH 8.8). Cells were harvested in PBS. Cytoplasmic fractions were extracted using buffer A with 1× protease inhibitors and 1 mM DTT. Nuclear pellets were cross-linked by 1.1% formaldehyde in buffer B with 1× protease inhibitors and 1 mM DTT; washed; and lysed in lysis buffer 3 with 1× protease inhibitors, 1 mM DTT, and 1 mM PMSF. The fixed and lysed nuclear extract was sonicated with Bioruptor® Pico (Diagenode) 10 times for 15 s each , with 45-s intervals. Chromatin was added to Dynabeads™ (Life Technologies) prebound with 4 µg of antibodies. For UTX chromatin immunoprecipitation (ChIP), chromatin and antibodies were incubated overnight, followed by 4 h of recapture with Dynabeads™ the next day. After incubation, beads were washed and immunoprecipitates were eluted. DNA from eluates was recovered by the GeneJET FFPE DNA purification kit (ThermoFisher Scientific, #K0882). ChIP-qPCR signals were calculated as the percentage of input. DNA libraries were generated using the NEBNext™ Ultra DNA Library Prep kit (NEB, #E7370S) and sequenced at the St. Jude Hartwell Center.