GSM1541514: yeast wild-type, 30 min H2O2, polysomes; Saccharomyces cerevisiae; OTHER
RNA isolated from yeast polysome or monosome fractions was purified using Trizol (Life Technologies), DNase I (Thermo Scientific) digested for 1 h, phenol-chloroform extracted, isopropanol precipitated and dissolved in 20 µl water. For C. elegans ~200 whole young adult hermaphrodites were washed 3x in PBS and lysed directly in Trizol by 30 cycles of sonication (30 s on, 30 s off) in a Bioruptor (Diagenode). The EZ RNA Methylation Kit (Zymo Research) was used for bisulfite treatment and purification of converted RNA according to the instructions provided by the manufacturer. 400 ng RNA were reverse-transcribed using Super Script III reverse transcriptase (Life Technologies) and gene-specific reverse primers (see below) for 50 min following the manufacturer’s instructions. 5 µl cDNA was subjected to the following touchdown-PCR program in a 25 µl reaction with KAPA2G Robust Polymerase and Buffer A (Kapa Biosystems):  95°C  3 min  95°C  30 s*  55-45°C 30s*  *20 cycles, -0.5°C annealing per cycle   72°C  1 min*  95°C  30 s**  45°C  30 s**  **35 cycles  72°C  1 min**  72°C  5 min  PCR products were gel-purified using PCR- and Gel Extraction Kit (Favorgen) and eluded in 30 µl. 5 µl were subjected to a second round of PCR as above using barcoded primers and only 30 cycles in the second cycling step (**). Up to 24 barcoded samples were pooled to equal molarity and subjected to IonTorrent sequencing using a 314 Torrent Sequencing Chip Kit (Life Technologies).    Primer sequences (without barcode):  SC-25S-rRNA_bisulfite_sense: TGGTTAGAAAGTGATGTT  SC-25S-rRNA_bisulfite_as: TCTCATTAATCCATTCA  CE-28S-rRNA_bisulfite_sense: TGAATGTTAAATTGTAGTAATT  CE-28S-rRNA_bisulfite_as: TCTCGTTAATCCATTCA    For barcode sequences see "barcode" field in the SAMPLES section