Cfx98 multi color system

Real-time qPCR was carried out in a CFX98 multi-color system (Bio-Rad) using SYBR Green Supermix as described previously (Mohan et al., 2015) using total RNA reverse-transcribed with the RT-PCR kit (Bio-Rad). Single-product amplification was confirmed by melting curve analysis, and primer efficiency was near 100% in all experiments. Quantifications are expressed in arbitrary units, and target mRNA abundance was normalized to the expression of GAPDH with the Pfaffl method. All real-time qPCR results are representative of at least three independent experiments (n > 3). For real-time qPCR of animal tissues, at least 5 animals were used (p < 0.05).

RIP experiments were carried out after cross-linking proteins and RNA in HEK293 cells with 1% formaldehyde using specific antibodies against Star-PAP, PAP⍺, RBM10, PIPKI⍺, and Pol II as described previously (Kandala et al., 2016) . For quantitative RIP, the immunoprecipitated RNA samples and input RNA were quantified using the CFX98 multi-color system (Bio-Rad) as described above. Values from each sample were corrected using reactions lacking reverse transcriptase. Quantifications are expressed in arbitrary units, and immunoglobulin G (IgG) immunoprecipitation product levels were used as controls for normalization of the abundance of the target messages. The quantitative association was then expressed relative to the input RNA signal as described previously (Selth et al., 2009) using the method of Pfaffl (Pfaffl, 2001) .