Chip sequencing sample preparation kit

Cells were grown in either normoxia or 1% O₂ induced hypoxia. Cells were cross-linked by adding formaldehyde to a final concentration of 1% at room temperature. Crosslinking was stopped by adding glycine with shaking. Cells were collected by centrifugation and washed three times before resuspending in PBS. Cell pellets were resuspended in cell lysis buffer (5 mM PIPES pH8.0/85 mM KCl/0.5% NP-40) containing protease inhibitors (1 µg/ml Aprotinin, 1 µg/ml Leupeptin, 1µ/ml Pepstatin, and 1 mM PMSF). Cells were incubated on ice and homogenized with several strokes using a douncer. Nuclei were collected by centrifugation and lysed by adding nuclear lysis buffer (50 mM Tris, Ph8.0/10 mM EDTA/1% SDS containing same protease inhibitors). After nuclear lysis, chromatin were fragmented to an average size of 300–400 bp with a Branson sonifier 250 with a microtip and cooling on ice. Cell debris was cleared by centrifugation and ChIP library preparation and adaptor ligation was performed using Illumina ChIP-sequencing sample preparation kit according to manufacturer protocol. ChIP-sequencing was performed at University of Washington sequencing core (St. Louis, MO). The data was analyzed using CLC Bio software (Qiagen Inc Germantown, MD).