title: GSM2652682: chromatinRNA_KDMot1Ino80_replicate1; Mus musculus; RNA-Seq
library_construction_protocol: For nascent-RNA, cells 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 ml of ice-cold 1× PBS/1 mM EDTA. The nuclear pellet was resuspended in 100 µl of prechilled 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 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 mix was vortexed vigorously for 2 seconds. The sample was incubated for 2 min on ice, and then centrifuged for 2 min at 4°C and 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 manual. For nascent-RNA-Seq, cells 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 ml of ice-cold 1× PBS/1 mM EDTA. The nuclear pellet was resuspended in 100 µl of prechilled 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 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 mix was vortexed vigorously for 2 seconds. The sample was incubated for 2 min on ice, and then centrifuged for 2 min at 4°C and 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 manual.
attributes.tag: GEO Accession
attributes.value: GSM2652682