Intergenerational hormesis is regulated by 18S rRNA methyltransferases DIMT-1 and BUD-23
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These are the raw files for SCARLET and primer extension assays.
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Primer extension and rRNA analysis 5 µg total RNA extracted using the TRI reagent (Invitrogen) was resolved on a denaturing agarose gel during 18h30 at 65V, stained with ethidium bromide and processed for northern blotting with probe LD2648 (5’-CACTCAACTGACCGTGAAGCCAGTCG-3’) or LD2649 (5’-GGACAAGATCAGTATGCCGAGACGCG-3’) (Bar et al., 2016) as in (Heissenberger et al., 2020). Primer extension was performed with primer LD4728 (5’-GACCGTGAAGCCAGTCGAGCATC-3’) on 5 µg total RNA as in (Zorbas et al., 2015). The northern blot and primer extension assays were exposed to Fuji imaging plates (Fujifilm) and quantification was performed on a phosphorimager (FLA-7000; Fujifilm) using the MultiGauge software (Fujifilm, v 3.1). The signals corresponding to the mature rRNAs were quantified using the chemidoc Image Lab software (v 4.0). Site-specific cleavage and radioactive-labeling followed by ligation assisted extraction and thin-layer chromatography (SCARLET) SCARLET assays were performed as in (Liu et al., 2013). Briefly, in the first step 18S rRNA was subjected to RNAse H site-specific cleavage directed by 2’-O-methyl RNA-DNA chimeras with the following sequences; C. elegans 18S rRNA G1531 chimeric oligo: 5’- mGmGmCmAmUmUmCCTCGmUmUmUmAmAmGmG-3’, C. elegans 18S rRNA A1735 chimeric oligo: 5’- mGmCmAmGmGmUmUCACCmUmAmCmAmGmCmU-3’, C. elegans 18S rRNA A1736 chimeric oligo: 5’- mUmGmCmAmGmGmUTCACmCmUmAmCmAmGmC-3’, H. sapiens 18S rRNA G1639 chimeric oligo: 5’- mGmGmAmAmUmUmCCTCGmUmUmCmAmUmGmG-3’, H. sapiens 18S rRNA A1850 chimeric oligo: 5’- mGmCmAmGmGmUmUCACCmUmAmCmGmGmAmA-3’. 200 ng of gel purified 18S rRNA was mixed with 5 pmoles chimeric oligo in 30 mM Tris-HCL, pH=7.5 in a total volume of 5 μl. The resulting mixture was heated for 3 min at 95°C followed by cooling to RT for 3 min. RNAse H (5 Units, NEB), rSAP (1 Unit, NEB) and RNasin (20 units, Promega) were added in a total volume of 10 μl in 1X T4 PNK buffer (NEB) and the mixture was incubated for 1 hr at 44°C, followed by heat inactivation for 5 min at 75°C. Radioactive end-labeling was performed with the addition of T4 PNK (20 Units, NEB) and 2 μl [γ-32P]ATP (6000Ci/mmol) at 37°C for 1 hr in a total volume of 15 μl in 1X T4 PNK buffer, followed by heat inactivation for 5 min at 75°C. The free [γ-32P]ATP was removed by the use of Bio-Spin 6 column (Biorad) according to the manufacturer’s instructions. The radioactive labeled 18S fragments were subjected to splint ligation by the addition of 5 pmoles splint oligo and 5 pmoles of 116-mer ssDNA oligo