GSM8121380: MOZ-TIF2 LSK cells, WM-1119 1h 1 uM treated, rep2; Mus musculus; RNA-Seq
To extract RNA, approximately 10 million cells were harvested and nuclei were extracted with nuclear extraction buffer (10% Sucrose, 20 mM HEPES pH 7.6, 10 mM NaCl, 3 mM MgCl2, 0.5% Triton X-100) supplemented with SUPERaseIN (Invitrogen AM2696). Nuclei were resuspended in 160 uL CF buffer (10 mM Tris pH 8, 100 mM NaCl, 25 mM EDTA) supplemented with SUPERaseIN. 1 mL fresh NUN buffer (1.5 M Urea, 20 mM HEPES, 300 mM NaCl, 7.5 mM MgCl2, 0.2 mM EDTA, 1% NP-40) was immediately added to lyse nuclei. Samples were incubated on ice for 10 minutes to precipitate genomic DNA, histones, engaged RNA PolII, and nascent transcripts. Samples were centrifuged at 20,000xg for 10 minutes and pellets were washed twice with NUN buffer. Pellets were resuspended in 700 uL CF buffer supplemented with 1 mg/ml Proteinase K and 1% SDS and incubated for 1 hour at 55oC to remove protein. Nucleic acids were isolated with phenol chloroform extraction. DNA was removed with Turbo DNAse (Invitrogen AM2238). RNA was extracted with Phenol:Chloroform:Isoamyl alcohol. To remove residual DNA, a second DNAse treatment was performed Sequencing libraries were generated using the NEBNext Ultra II Directional RNA Library Preparation Kit (New England Biolabs E7760).