PIP2-TAP constructed from CEN.PK 113-5D by adding a C-terminal CBP-ProtA tag to PIP2p
carbon source
0.75% (w/v) glucose
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Formaldehyde with a final concentration of 1% (w/v) and distilled water were added to the culture to cross-link protein-DNA complexes with OD600 of 1.0 and a total volume of 100 ml. Cross-linking was performed for 12 min at room temperature with shaking and quenched by adding 2.5 M glycine to a final concentration of 125 mM. After 5 min, cells were washed twice with 20 ml cold TBS (10 mM Tris-HCl, 150 mM NaCl, pH7.5) and frozen with liquid nitrogen. For ChIP-exo, cells were disrupted with glass beads on FastPrep 24 (MP Biomedicals) and the crude cell lysate was sonicated to shear chromatin on Branson digital sonifier 250 (Branson Ultrasonics). After centrifugation, the supernatant containing chromatin fragments was applied to IgG Sepharose 6 Fast Flow beads (GE Healthcare) for immunoprecipitation at 4°C with gentle rocking overnight. NEBNext End Repair Module, NEBNextdA-Tailing Module, NEBNext Quick Ligation Module, PreCR Repair Mix, Lambda exonuclease and RecJf (all from New England Biolabs) were used for on-bead end repair, 3´-dA DNA tailing, first adaptor ligation, nick filling-in and chromatin trimming, respectively. The first adaptors contain unique 6 bp-index sequences. To elute and reverse cross-link the bound complexes, TE buffer containing 1% SDS was added to the beads and samples were incubated overnight at 65°C. After protease K (Thermo Scientific) digestion and DNA extraction with phenol/chloroform/isoamyl alcohol (Amresco, US), the single-strand DNAs were subjected to primer extension using phi29 DNA polymerase (New England Biolabs). The products were dA-tailed and ligated with the second adapter using the same reagents as in on-bead reactions, and then amplified by PCR for 20-22 cycles using Phusion High-Fidelity DNA Polymerase (New England Biolabs). GeneRead Size Selection Kit (QIAGEN) was used to purify DNA before and after the second dA-tailing, second adapter ligation and PCR. The DNA samples prepared with ChIP-exo contain adaptors and are ready for direct sequencing with Illumina sequencer. The samples were pooled together in equimolar amounts before sequencing.