title: GSM2060956: B6_BMDM_IRF1_IFNg_0.5h; Mus musculus; ChIP-Seq
library_construction_protocol: Twenty million BMDMs per ChIP were crosslinked for 10 min at room temperature with 1% formaldehyde added in culture medium. Crosslink was stopped with ice-cold PBS containing 0.125M glycine for 5min. Nuclei were prepared by sequential incubation on ice for 5min in buffer A [10mM Tris-HCl (pH 8), 10mM EDTA, 0.25% Triton X-100], and for 30min in buffer B [10mM Tris-HCl (pH 8), 1mM EDTA, 200mM NaCl] (all buffers include proteases inhibitors). Nuclei were resuspended in sonication buffer [10mM Tris-HCl (pH 8), 1mM EDTA, 0.5% SDS, 0.5% Triton X-100, 0.05% NaDOC, 140mM NaCl] and sonicated with a Branson Digital Sonifier (Branson Ultrasonics) to an average size of 250bp. Sonicated chromatin was incubated overnight on a rotating platform at 4°C with a mixture of 20μl Protein A and 20μl Protein G Dynabeads (Invitrogen) pre-bound with 6μg of control IgG (sc-2028) or IRF8 (sc-6058), IRF1 (sc-640), PU.1 (sc-352) antibodies from Santa Cruz Biotechnologies or 3μg of H3 (ab1791), H3K27Ac (ab4729) from Abcam. Immune complexes were washed sequentially for 2min at room temperature with 1ml of the following buffers: Wash B [1% Triton X-100, 0.1% SDS, 150mM NaCl, 2mM EDTA, 20mM Tris-HCl pH 8]; Wash C [1% Triton X-100, 0.1% SDS, 500mM NaCl, 2mM EDTA, 20mM Tris-HCl pH 8), Wash D (1% NP-40, 250mM LiCl, 1mM EDTA, 10mM Tris-HCl pH 8]; and TEN buffer [50mM NaCl, 10mM Tris-HCl pH 8, 1mM EDTA]. After de-crosslinking by overnight incubation at 65°C, the DNA was purified with QIAquick PCR purification columns following manufacturer’s instructions (Qiagen). ChIP-seq libraries were prepared using Illumina TruSeq kit and sequenced on a HiSeq 2000 in paired-end 50bp configuration following manufacturer’s recommendations.
identifiers.namespace: GEO
identifiers.id: GSM2060956