GSM2702916: Subcellular RNA-seq Chromatin serum-starved Rep 2; Mus musculus; RNA-Seq
Subcellular fractionation and RNA preparation were performed essentially as described (Bhatt et al. 2012) with minor modifications. 10^6 cells were serum starved and stimulated with TPO as above. Cells were collected by centrifugation and washed once with PBS. The cell pellet was resuspended in ice-cold NP-40 lysis buffer (10 mM Tris-HCl pH 7.5, 0.15% NP40, 150 mM NaCl). The lysate was then layered on 2.5 volumes of a sucrose buffer and centrifuged for 10 min at 13,000 rpm at 4ºC. The supernatant (cytoplasmic fraction) was collected and added 3.5X volumes of RLT Buffer (Qiagen). The nuclei pellet was gently rinsed with ice-cold 1X PBS and resuspended in 200μl 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) by gently flicking the tube. 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 and gently vortexed twice for 2 sec, incubated for 2 min on ice, and then centrifuged at 13,000 rpm for 2 min at 4°C. The supernatant (nucleoplasmic fraction) was collected and added 3.5X volumes of RLT (Qiagen). The chromatin pellet was gently rinsed with cold 1X PBS and then dissolved in 500 μl TRIzol (Invitrogen). RNA purification from RLT-dissolved samples was performed using RNeasy columns (Qiagen). Chromatin-associated RNA was purified using Direct-zol (Zymo Research). Libraries were prepared according to Illumina's instructions accompanying the DNA and RNA Sample Kit. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on a HiSeq4000 sequencer.