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. Author manuscript; available in PMC: 2021 Jul 1.
Published in final edited form as: Nat Protoc. 2020 Nov 18;16(1):27–60. doi: 10.1038/s41596-020-00404-1

Figure 4. eRIC quality controls.

Figure 4

Poly(A) RNA and associated RBPs were purified from proliferating human leukemia cells (Jurkat) with eRIC and analyzed as follows.a) Representative bioanalyzer profiles of RNA extracted from input material (top), eRIC pre-eluates (middle), and eRIC heat-eluted samples (bottom). rRNAs, tRNAs and small RNAs are predominant in inputs and still present in pre-eluates. eRIC heat-eluted samples display a strong enrichment of RNA species with a size range characteristic of mRNAs. Both 28S rRNA and the small RNAs are undetectable in the heat eluates, although a residual amount of 18S rRNA is consistently observed. [FU] fluorescent units, [nt] length of RNA in number of nucleotides.b) eRIC was performed on Jurkat cells incubated for 6 h with 0.5 mM of the a-ketoglutarate antagonist dimethyloxalylglycine (DMOG) or vehicle (DMSO). Aliquots of the RNase-eluted samples and inputs were analyzed by SDS-PAGE and silver staining. Except for the RNase A and T1 bands (~15 KDa), proteins are only detected in eRIC eluates if cells were UV-irradiated. Protein patterns in untreated and DMOG-treated samples are virtually indistinguishable, which agrees with the proteomic results that revealed a very limited RBP pool (61 proteins out of 721) responding to DMOG14.