Instrument: Illumina Genome Analyzer II
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Kasumi-1 cells growing in log phase were fixed with 1% formaldehyde for 10 minutes at room temperature and subsequently quenched with 0.25 M glycine. After washes in PBS, cells were flash frozen in liquid nitrogen. Thawed pellets were resuspended in Buffer A (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) and rotated at 4ºC for 10 minutes. Following centrifugation, pellets were resuspended in Buffer B (10 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 1 mM EGTA) and rotated at 4ºC for 10 minutes. Pellets from centrifuged samples were resuspended in Buffer C (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine). Samples were aliquoted, 3 ml per tube, and sonicated to a fragment size of 100-500 base pairs using a 3.2 mm sonication probe (QSonica, Newtown, CT). Sheared chromatin (from approximately 30 million cells) was immunoprecipitated with antibodies overnight. All ChIP experiments included either normal goat or rabbit IgG (EMD Millipore, Billerica, MA) as controls. Antibody-lysate complexes were mixed with Protein G Dynabeads (Life Technologies, Grand Island, NY) for 2 hours. For AML1, ETO, H3K4me3, and H3K27me3 immunoprecipitations, beads were washed once with IP buffer, 3 times with RIPA buffer (50 mM HEPES, 500 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.25% sodium deoxycholate), once with PBS, and once with TE buffer. For N-CoR and p300 pulldowns, beads were washed once with IP buffer, once with high salt buffer (2 mM EDTA, 20 mM Tris-HCl, 500 mM NaCl), 1 time with RIPA buffer (50 mM HEPES, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.25% sodium deoxycholate), once with PBS, and once with TE buffer. Protein-DNA complexes were extracted from beads at 37ºC in elution buffer (10 mM EDTA, 50 mM Tris-HCl, 1% SDS). For reverse crosslinking, supernatants from centrifuged samples were rotated overnight at 60ºC. RNAse and proteinase K treated samples were extracted with phenol:chloroform:isoamyl alcohol. Precipitated DNA was resuspended in 10 mM Tris-HCl, quantified with a Qubit fluorometer (Life Technologies, Grand Island, NY). The Illumina protocol (Illumina, Inc., San Diego, CA) for ChIP-seq library generation was used with slight modifications. Approximately 5-10 ng chromatin was end-repaired (EpiCentre Biotechnologies, Madison, WI). Material was then A-tailed and ligated with adapters for single end deep sequencing (Illumina, Inc., San Diego, CA). Adapter modified DNA was size-selected, 300-400 base pair (bp) range, and then amplified using the Phusion polymerase (New England Biolabs, Ipswich, MA). Amplified ChIP libraries were size selected and sequenced on an Illumina GAIIx Genome Analyzer (Illumina, Inc., San Diego, CA) at the UMass Medical School Deep Sequencing Core Facility (Worcester, MA).