Instrument: Ion Torrent Proton
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Cells were crosslinked with 1% formaldehyde in culture medium for 30 min at room temperature followed by quenching with 0.125 M glycine for 5 min. Cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (50mM Hepes KOH pH 7.5, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate) using Zirconia beads. Crosslinked chromatin was sheared to an average size of 500 bp by 6X 15-second pulses using a Biorupter sonicator. The lysate was then centrifuged to remove cell debris. The chromatin fraction was incubated with Dynabeads protein G beads (Invitrogen, cat no 10003D) coated with anti-Flag antibody (M2-antiflag, Sigma) overnight at 40C. The immune complexes were washed with the following buffers 2X; Chip-lysis buffer, high-salt lysis buffer (Chip-lysis buffer + 360mM NaCl), Chip-wash buffer (250 mM LiCl, 10 mM Tris pH8.0, 0.5% Na deoxycholate, 0.5% NP40, 1mM EDTA) and 1x TE (20 mM Tris pH8.0, 2 mM EDTA). The protein-DNA complexes were eluted from the beads in 250 µl elution buffer (1% SDS, 50 mM Tris pH8.0, 10mM EDTA) at 650C for 20 min followed by the addition of proteinase K to 500 µg/ml and overnight incubation at 650C. Input DNA was isolated from sheared chromatin input (1/100 of the material used for ChIP). IP and Input ChIP-seq libraries were prepared according to the manufacturer protocols for the Ion Proton sequencer (Thermo Fisher Scientific/Life Technologies). Briefly 10ng for ChIP DNA was end repaired and adapter ligated using the KAPA Library Preparation Kit for Ion Torrent™ (KAPABIOSYSTEMS, inc) and adapter barcode Kapa Barcode Adaptors 9-24 . After adapter ligation, each sample was size selected using AMPure XP Bead (Beckman Coulter, inc). An amplification reaction was set up in a final volume of 50ul . A SPRI cleanup with a 1.5X Bead:DNA ratio was performed post amplification and final libraries were eluted in 35ul. Libraries were quantified on Qubit fluorometer with HS DNA (Thermo Fisher Scientific/Life Technologies) and checked for size on an Agilent Bioanalyzer with HS DNA kit (Agilent, Santa Clara, CA). Each size selected library was diluted according to the final concentration of 11 pM and clonally amplified using the Ion Proton™ Hi‑Q™Template Kit (Thermo Fisher Scientific/Life Technologies) with IonOneTouch 2 instrument (Thermo Fisher Scientific/Life Technologies). After emulsion PCR, DNA positive ISPs were recovered and enriched according to standard protocols with the IonOneTouch ES Instrument (Thermo Fisher Scientific/Life Technologies). A sequencing primer was annealed to DNA positive ISPs and the sequencing polymerase bound, prior to loading of ISPs into Ion P1 sequencing chips. Sequencing of the samples was conducted according to the Ion Proton™ Hi-Q Sequencing Kit Protocol. One P1 sequencing chips with 6 libraries were loaded and run on an Ion Proton sequencer.