ChIP-seq of PAX8 and H3K27ac

Chromatin immunoprecipitation sequencing (ChIP-seq) was performed based on the methods of Schmidt et al. [36 (link)]. Cells were fixed in 1% formaldehyde for 10 minutes, and quenched with glycine. Cells were harvested, lysed in a sarkosyl-containing buffer, and sonicated using the Covaris E220evolution Focused-Ultrasonicator. 10 μg of an antibody raised against PAX8 (NBP1-32440, Novus) or 5 μg of an antibody raised against lysine 27 acetylated histone 3 (H3K27ac) (C15410196, Diagenode) was incubated with 100 μg and 4ug, respectively, of chromatin at 4°C overnight. Blocked magnetic Dynabeads (Life Technologies) were then added to the antibody-lysate conjugates and incubated at 4°C for 4 hours with rotation. Beads were then washed with RIPA buffer and treated with RNase and proteinase K (both Qiagen). DNA was eluted from the beads in Tris-EDTA buffer and cleaned up using the QIAquick PCR Purification kit (QIAgen). For each cell line two independent immunoprecipitations and one input sample were submitted for next-generation sequencing at the USC Epigenome Core Facility.