GSM1261037: GM12750_chromatin_RNA; Homo sapiens; RNA-Seq
For chromatin-associated nascent RNA-seq, cultured B cells were treated with cell fractionation buffer from the PARIS kit (Ambion) following manufacturer’s protocol to obtain a nuclei pellet.  Chromatin fraction was extract from this pellet as previously described (Pandya-Jones and Black, RNA, 15, 1896, 2009).  Briefly, the nuclei pellet was washed with ice-cold 1× PBS containg 1 mM EDTA, then resuspended in a ice-cold glycerol buffer (20 mM Tris-HCl, pH 7.9, 75 mM NaCl, 0.5 mM EDTA, 0.85 mM DTT, 0.125 mM PMSF, 50% glycerol). An equal volume of ice-cold nuclei lysis buffer (10 mM HEPES, pH 7.6, 1 mM DTT, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1 M UREA, 1% NP-40) was added, the tube was gently vortexed, incubated for 2 min on ice, and then centrifuged for 2 min, 4°C at 14,000 rpm.  The supernatant was removed and the chromatin pellet was gently washed with ice-cold 1× PBS containg 1 mM.  Chromatin RNA was prepared from this pellet using TRI Reagent RT (Molecular Research Center). This RNA was further purified using the RNeasy Mini protocol (Qiagen) with on-column DNase digestion.  After being shown to be free of genomic DNA, the chromatin RNA was sequenced on HiSeq 2000 as described above. Libraries were prepared following Illumina Directional mRNA-seq sample preparation protocol.
GEO Accession
GSM1261037