Rna century plus marker template and maxiscript t7transcription kit

1 μg of total RNA from HEK293T cells was combined with 1 ng total RNA from S. pombe and enriched with MTS biotin exactly as in (29 (link)) or with MTS-resin as above. Enriched samples were assayed by RT-qPCR as above and fold enrichment was calculated as

An RNA ladder of 100–1000 nt was transcribed in vitro using the RNA Century Plus Marker Template and Maxiscript T7 transcription kit (Invitrogen) using Cy5-CTP at a ratio of 1:1 Cy5-CTP:CTP for downstream visualization, with the option of adding s⁴UTP (TriLink Biotechnologies) at a ratio of 1:1 s⁴UTP:UTP to the reaction. After the reaction, template DNA was digested with Turbo DNase (Thermo Fisher). Enzymes were removed by phenol-chloroform extraction and the RNA was purified using the RNeasy Mini Kit (QIAGEN) with the following changes: β-mercaptoethanol was added to wash buffer RPE to a final concentration of 1%. 400 ng of RNA ladder was enriched with MTS-resin following the protocol described above, or with HPDP-biotin and MTS-biotin following protocols in (18 (link)). Enriched samples were separated on a 5% urea-PAGE gel, visualized by Typhoon fluorescence imager (GE), and bands were quantified using ImageJ software.