RNA-fractionation sequencing

To identify potential RBM33 substrates, we performed whole transcriptome sequencing (RNA-seq) on nuclear and cytoplasmic RNA from RBM33 wild-type and knockout cells 400 000 WT, RBM33 KO-1, or RBM33 KO-2 HCT116 cells were seeded in 6-well plates. For each replicate, 48 hours later, one well was subjected to total RNA extraction and one well to nuclear-cytoplasmic fractionationFractionation was performed as described previously (Lee et al., 2016). Briefly, cells were collected by trypsinization and lysed in 175 µL RLN1 buffer [50 mM Tris-HCl pH 8, 140 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40, and 1:1000 SUPERase In RNase inhibitor (Thermo Fisher Scientific)]. Following a 5-minute incubation on ice, the sample was centrifuged at 300 g for 2 minutes. The supernatant, representing the cytoplasmic fraction, was collected and centrifuged at 16000 g for 5 minutes to clear debris. The initial pellet, representing the nuclear fraction, was gently resuspended in 500 µL RLN1 and centrifuged at 1000 g for 5 minutes to wash. The pellet was then resuspended in 175 µL RLN2 buffer [50 mM Tris-HCl pH 8, 500 mM NaCl, 1.5 mM MgCl2, 0.5% NP-40, and 1:1000 SUPERase In RNase inhibitor (Thermo Fisher Scientific)]. RNA was extracted from each of these fractions by Trizol in combination with the miRNeasy mini kit (Qiagen) with an on-column DNase digestion.