GSM3263581: Nascent_RNAseq_siEsrrb_R2; Mus musculus; RNA-Seq
mESCs were harvested, washed with DPBS and lysed in 200 μl of ice-cold lysis buffer (10 mM Tris-HCl pH 7.5, 0.1% NP40, 150 mM NaCl). The cell lysate was gently layered over 500 μl of chilled sucrose cushion (24% RNAse-free sucrose in lysis buffer) in a new Eppendorf tube and centrifuged for 10 min at 4°C, 10,000xg. The supernatant (cytoplasmic fraction) was removed and the pellet (nuclei) was washed once with 200 μl of ice-cold 1 × PBS/1 mM EDTA. The nuclear pellet was resuspended in 100 μl of pre-chilled 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 gentle flicking of the tube. An equal volume (100 μl) of cold nuclear 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 mix was vortexed vigorously for 2 s. The sample was incubated for 2 min on ice, and then centrifuged for 2 min at 4°C, 10,000xg. The supernatant (nuclear fraction/nucleoplasm) was removed and the pellet (chromatin) was gently rinsed with ice-cold 1×PBS/1 mM EDTA. 1 ml of Trizol reagent was added to the chromatin and incubated for 30 min at 50oC to dissolve it. Nascent RNA was extracted following the manufacturer's instructions. Libraries were constructed using KAPA Stranded RNA-Seq Kits with RiboErase (HMR). Nascent RNA-seq libraries were constructed using KAPA Stranded RNA-Seq Kits with RiboErase (HMR).