GSM5399303: V65_nascent_RNAseq_Rep2; Mus musculus; RNA-Seq
Mouse ESCs were harvested at ~80% confluency and washed with DPBS. Cells were counted and 10 million cells were collected for subsequent steps. Half a million human BEAS-2B cells were mixed into the 10 million mouse ESCs as the spike-in. Cells were 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 a chilled sucrose cushion (24% RNase-free sucrose in lysis buffer) in a new Eppendorf tube and centrifuged for 10 min at 4°C at 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 at 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 50°C to dissolve it. Nascent RNA was extracted following the manufacturer's instructions. Libraries were constructed using KAPA Stranded RNA-Seq Kit with RiboErase (HMR).