Nuclear RNA-Seq of GH3 Cells
The nuclear RNA samples were obtained from a test intended for lariat and RNA secondary structures. Briefly, GH3 cells were untreated, treated with KCl (50 mM), or KCl (50 mM) plus 5-azaC (50 M), respectively in triplicate (three wells of cells). Upon 6h treatment, cells were collected and nuclear RNA extracted in a procedure modified from the nucleo-cytoplasmic fractionation protocol [12,53]: cells were lysed with cold 0.325% NP-40 buffer (10 mM Tris-Cl pH 7.5, 75 mM NaCl, 0.325% IGEPAL CA-630), centrifuged, and the nuclear pellet washed with ice-cold PBS plus 1 mM EDTA, resuspended and lysed in 0.65% NP40 buffer (10 mM Tris-Cl pH 7.5, 75 mM NaCl, 0.65% IGEPAL CA-630, and 10mM FeCl3 [54,55]) with 10 g/mL psoralen [56], chilled in an aluminum block on ice for 10min, and ultraviolet-irradiated (365 nm) for 20min., and extracted with the QIAquick Gel Extraction Kit (Qiagen, Cat.# 28706, Hilden, Germany) for nuclear RNA. Approximately 1 g of nuclear RNA from each sample of the three groups was reverse transcribed using random primers for NEB rRNA-depleted (HMR) stranded library and NovaSeq 6000 sequencing. On average, 42 million uniquely mapped reads (paired, 6.6, n = 12) were obtained for each sample.