Instrument: Illumina HiSeq 3000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: extract 1: For ChIP-exo, after treatment with 10 nm DHT or vehicle for 4 h, LNCaP cells were crosslinked with 1% formaldehyde for 10 min. Sonicated chromatin was enriched by immunoprecipatation with an anti-GATA2 antibody and T4 DNA polymerase, T4 PNK, and Klenow DNA Polymerase were used together for end polishing. The ligation step was performed with less reducing agent. Protein A Dynal magnetic beads were washed using modified RIPA buffer (50mM Tris-HCL pH 7.8, 1mM EDTA, 0.25% Na Deoxycholate, 1% NP-40, 0.5M LiCl). The library was amplified with only 10 or 12 PCR cycles, and prepared without gel-based size selection. extract2: For Mnase-ChIP-seq, chromatin was isolated from freshly cultured cells and Mono-nucleosomes with solubilized chromatin was achieved by MNase digestion at 37 °C, then immunoprecipitated with antibody-conjugated magnetic beads. DNA is phenol extracted and ethanol precipitated. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part#15023092). Briefly, DNA was end-repaired, phosphorylated ends were treated with Klenow fragment and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation, the DNA was size selected from an agarose gel and was PCR amplified with Illumina primers for 15 cycles. Libraries were sequenced on the Hi-seq 3000 following the manufacturer's protocols.