GSM4174051: TGFb2h, biological rep1 [Chromatin-enriched RNA-seq]; Mus musculus; RNA-Seq
Cells were resuspended in 400 μl cold cytoplasmic lysis buffer (0.15% NP-40, 10 mM Tris pH 7.5, 150 mM NaCl) and incubated on ice for 5 min. The lysates were layered onto 1 ml cold sucrose buffer (10 mM Tris pH7.5, 150 mM NaCl, 24% sucrose w/v), and centrifuged in microfuge tubes at 3500 g for 10 minutes. The nuclear pellets were gently resuspended into 250 μl cold glycerol buffer (20 mM Tris pH 7.9, 75 mM NaCl, 0.5 mM EDTA, 50% glycerol). An additional 250 μl of cold nuclei lysis buffer (20 mM HEPES pH 7.6, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1 M urea, 1% NP-40, 1 mM DTT) was added to the samples, followed by a pulsed vortexing and incubation on ice for 2 minutes. Samples were then spun in microfuge tubes for 2 min at 13000 g. 50 μl of cold PBS was added to the remaining chromatin pellet, and gently pipetted up and down over the pellet, followed by a brief vortex. Then RNA was extracted using TRIZOL and treated with TURBO DNA-free kit (AM1907, Invitrogen) for DNA removal according to manufacturer's instructions The libraries were prepared with the TruSeq Stranded TOTAL RNA kit (Illumina, San Diego, CA, USA) and the sequencing was performed with the HiSeq2000 1x50bp length
GEO
GSM4174051
GEO Accession
GSM4174051