Cation-exchange chromatography enriches for Argonaute- and Piwi-enclosed RNAs. (A) Phase contrast image of OSS cells illustrating their sheet-like morphology; under high density they also tend to form clumps. (B) pCp labeling of RNAs from total OSS RNA and various elutions from a HiTrap Q column. The mobility of the RNAs in the extract and elutions is slightly different from that of the synthetic markers owing to different salt concentrations of the loaded samples. Highly abundant 2S rRNA is visible in the input and ran slightly larger than its 30-nt sequence. In the flowthrough, ∼21–24-nt RNAs are visible, inferred to include miRNAs and siRNAs. In the 0.3 M salt elution, ∼24–28-nt RNAs are visible, inferred to represent the piRNA fraction. A heterogenous ladder of RNA fragments elutes with 1.0 M salt. Libraries were constructed from the flowthrough and 0.3 M salt elution.
