GSM2341297: ZHBTC4_NANOG_UNT_rep2 (ChIP-Seq); Mus musculus; ChIP-Seq
Chromatin immunoprecipitation was performed as described previously (Farcas et al., 2012), with minor modifications. Cells were fixed for 1 h in 2 mM DSG and 12.5 min in 1 % formaldehyde. Reactions were quenched by the addition of glycine to a final concentration of 125 µM. After cell lysis and chromatin extraction, chromatin was sonicated using a BioRuptor sonicator (Diagenode), followed by centrifugation at 16,000 × g for 20 min at 4°C, and used fresh or stored at -80°C. Chromatin was quantified by denaturing chromatin 1:10 0.1 M NaOH, and measuring DNA concentration by NanoDrop and 150 µg chromatin/IP was diluted ten-fold in ChIP dilution buffer (1% Triton-X100, 1 mM EDTA, 20 mM Tris-HCl (pH 8), 150 mM NaCl) prior to pre-clearing with prepared protein A magnetic Dynabeads (Invitrogen, Carlsbad, CA) which had been blocked for 1 h with 0.2 mg/mL BSA and 50 µg/mL yeast tRNA. Antibody-bound chromatin was isolated on protein A magnetic Dynabeads. ChIP washes were performed with low salt buffer (0.1 % SDS, 1 % Triton, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 150 mM NaCl), high salt buffer (0.1 % SDS, 1 % Triton, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), 500 mM NaCl), LiCl buffer (0.25 M LiCl, 1 % NP40, 1 % sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl (pH 8.1)) and TE buffer (x2 washes) (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). To prepare ChIP-seq material, ChIP DNA was eluted using 1 % SDS 100 mM NaHCO3, and cross-links reversed at 65°C in the presence of 200 mM NaCl. Samples were then treated with RNase and proteinase K before being purified with ChIP DNA Clean & Concentrator™ kit (Zymo, Irvine, CA). ChIP-seq libraries were prepared using the NEBNext Ultra DNA Library Prep Kit, and sequenced as 40bp paired-end reads on Illumina NextSeq500 platform. All ChIP-seq experiments were carried out in biological triplicate.
GEO Accession
GSM2341297