title: GSM1695192: H3K36me3 ChIP ccRCC2; Homo sapiens; ChIP-Seq
library_construction_protocol: Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. Nephrectomy samples underwent gross macrodissection to ensure that they contained >60% to 70% tumor, with verification by a mirror slide stained with hematoxylin-eosin. The frozen tissues were cut and divided into 50-mg aliquots and stored in 1.5-mL centrifuge tubes at -70°C. The frozen tissues were homogenized on ice for 15–30 seconds in 500 μL 1X PBS using a tissue grinder (ACTGene). Tissue homogenates were cross-linked to final 1% formaldehyde for 10 min and quenched with 125 mM glycine for 5 min at room temperature and washed once with TBS. The pellets were resuspended in cell lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.5% NP-40) and incubated on ice for 10 min. The lysates were divided into two aliquots and washed with MNase digestion buffer (20 mM Tris-HCl, pH 7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2). After resuspending in 500 μL MNase digestion buffer containing a proteinase inhibitor cocktails (ActiveMotif 37491), the lysates were incubated in the presence of 1,000 gel units of MNase (NEB M0247S) at 37 °C for 20 min with continuous mixing in a thermal mixer. After adding the same volume of sonication buffer (100 mM Tris-HCl, pH 8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% sodium deoxycholate), the lysates were sonicated for 15 min (30 seconds on / 30 seconds off) using Bioruptor Twin (Diagenode, model UCD-400) and centrifuged at 21,130 x g for 10 min. The cleared supernatant equivalent to 10–20 mg of tissue was incubated with 2 micrograms rabbit polyclonal anti-H3K36me3 antibody (Active Motif  61101) on a rocker overnight. After adding 30 microliters of protein G-agarose beads, the reactions were further incubated for 3 hours. The beads were extensively washed with ChIP buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), high salt buffer (50 mM Tris-HCl, pH8.1, 10 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), LiCl2 buffer (10 mM Tris-HCl, pH 8.0, 0.25 M LiCl2, 0.5% NP-40, 0.5% Sodium deoxycholate, 1 mM EDTA), and TE buffer. Bound chromatin were eluted and reverse-crosslinked at 65 °C overnight. DNAs were purified using Min-Elute PCR purification kit (Qiagen 28004) after the treatment of RNase A and proteinase K.
attributes.tag: GEO Accession
attributes.value: GSM1695192