title: GSM1175358: PU.1Ad-PU.1; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were cross-linked in 1% formaldehyde for 10 min, followed by quenching with 1/20 volume of 2.5 M glycine solution, and two washes with 1× PBS. Nuclei were extracted by brief probe sonication in hypotonic lysis buffer (20mM Hepes, 3mM MgCl2, 250mM sucrose, 0.2% NP-40, 3mM beta-mercaptoethanol) followed by three washes of nuclei in this buffer. Nuclei were lysed in ChIP SDS lysis buffer (50 mM HEPES, 1% SDS, 10 mM EDTA), followed by sonication with a Bioruptor (Diagenode) - 2x cycles of 30" ON, 30" OFF x 7.5'. 5% of the extract was removed for input DNA and the remainder was used for ChIP. Extracts were diluted in ChIP dilution buffer (50 mM Hepes/NaOH at ph 7.5, 155 mM NaCl, 1.1% Triton X-100, 0.11% Na-deoxycholate, 1 mM PMSF, Complete protease inhibitor tablet) and incubated with 10ug of Ab for 16h at 4°C. Antibody-antigen complexes were captured on ProteinA-sepharose beads (GE, CL-4B) for four hours followed by a series of bead washing steps : 1x 1' with ChIP dilution buffer, 1x 5' with ChIP dilution buffer, 1x 5' with ChIP dilution buffer +500mM NaCl, 1x 5' with ChIP wash buffer (10mM Tris-HCl pH 8, 1mM EDTA, 250mM LiCl, 0.5% NP-40, 0.5% NaDOC). Elution from beads and cross-link reversal of ChIPs and inputs was performed overnight at 65°C in ChIP elution buffer (50mM Tris-HCl pH 8, 10mM EDTA, 1% SDS). Proteins were digested with Proteinase K and DNA was isolated using phenol/chloroform/isoamyl alcohol. Libraries for each ChIP-Seq experiment were prepared as per Illumina's instructions (http://www.illumina.com). Briefly, DNA fragments were blunted, phosphorylated, and ligated to Illumina library adapters. Size selection was performed using gel electrophoresis by excising DNA fragments at 200 ± 50 base pairs. Following gel purification, PCR amplification was performed (30 s at 98°C; [10 s at 98°C, 30 s at 65°C, 30 s at 72°C] × 18 cycles; 5 min at 72°C). Amplified material was evaluated on an Agilent 2100 Bioanalyzer to ensure proper size selection.