GSM2042669: pol2s2p_norm3; Mus musculus; ChIP-Seq
Small liver pieces were incubated for 10 min at room temperature in 1× phosphate-buffered saline (PBS) containing 1% formaldehyde. The crosslinking reaction was quenched by adding glycine to a final concentration of 0.125 M. After a wash in 1× PBS, liver pieces were homogenized in lysis buffer and incubated for 10 min on ice. Nuclei were pelleted and resuspended in 1× TE buffer. Nuclei were sonicated using a Bioruptor (Diagenode, Liege, Belgium). Chromatin samples were diluted with 2× RIPA buffer to a final 1× concentration. IPs were performed with primary antibody and Dynabeads Protein A complex overnight at 4°C. Immune complexes were washed twice for 10 min in each of the following buffers: 1× RIPA buffer, 1× RIPA + 0.3 M NaCl, LiCl buffer, 1× TE + 0.2% Triton X-100, 1× TE. Then, immune complexes were in 100 μl 1× TE, protease K, and 0.3% sodium dodecyl sulfate (SDS) and incubated at 65°C overnight. Eluted DNA was purified using a QIAGEN PCR purification kit. Buffers were comprised of the following: lysis buffer (10 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1× TE; 10 mM Tris-HCl [pH 7.5], and 1 mM EDTA), 1× RIPA (1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and 10 mM Tris [pH 7.5], 1 mM EDTA), LiCl buffer (0.25 M LiCl, 0.5% NP40, and 0.5% sodium deoxycholate). Every buffer included 1× protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN). The following antibodies were used: H3Ace (06-599, Upstate/Millipore, Temecula, CA), H3K36me3 (ab9050, abcam), H3K27me3 (07-449, Upstate/Millipore, Temecula, CA), H3K4me3 (07-473, Upstate/Millipore, Temecula, CA), RNA polymerase II (ab5131, abcam). Illumina ChIP library prep (Part # 11251892 Rev. A)
GEO Accession
GSM2042669