GSM4219507: B6_Th1_Zbtb7b; Mus musculus; ChIP-Seq
Ten million Th1 cells 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 5μg of anti-ThPOK (sc-79127X) antibody from Santa Cruz Biotechnologies. 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 ChIP and Input DNA was purified with QIAquick PCR purification columns following manufacturer's instructions (Qiagen). The ChIP-seq libraries were prepared using the Kapa ChIP Library kit (Kapa Biosystems) and sequenced on a HiSeq 2500 (Illumina) using a 50bp paired-end configuration, with sonicated input DNA as control.
GEO Accession
GSM4219507