GSM5269325: HEK293 FS mutant NUP98-HOXA9 (HA) HA IP; Homo sapiens; ChIP-Seq
Fixed cells were sonicated in a Bioruptor sonication device for 45 cycles at a high-energy setting to enrich the sonicated DNA to be ~300bp as described in our method. The sonicated samples were incubated with the indicated antibodies pre-bound to the protein-G/A beads for overnight. Across 293 cell samples with GFP-tagged chimera, the cleared cell lysates from sonicated nuclei were added with the same fraction of the sonicated and cleared chromatin of Drosophila S2 cells (see our Method), and then protein-DNA complexes were isolated with antibodies against GFP and those against Drosophila-specific histone H2Av (Active Motif cat# 39715). Beads were then washed with various washing buffers (please see our method). Bead-bound DNA was eluted and subject to reverse crosslinking and sequential treatments of RNase and protease K. Finally, ChIP DNA was purified with a PCR cleanup kit (Qiagen) and used for library construction. Multiplexed ChIP-seq sequencing libraries were prepared using the NEBNext Ultra II kit (NEB, #E7645L) following the manufacture's instruction. ChIP-seq libraries were pooled together and sequenced on a Nextseq 550 system using a Nextseq 550 High Output Kit v2.5 or sent to UNC High-Throughput Sequencing Facility (HTSF) for sequencing by using an Illuminia Hiseq 2500/4000 system.