GSM4969773: disome-seq_negative_control; Saccharomyces cerevisiae; OTHER
Ribosomes were extracted with the polysome lysis buffer (PLB), which contained 200 mM Tris-HCl (pH 8.0), 200 mM KCl, 35 mM MgCl2, 1% (v/v) Triton X-100, 5 mM DTT, and 50 µg/mL cycloheximide. 50000 units (A260) of ribosome dissolved in the PLB buffer were treated with 750 U RNase I (Ambion, AM2294) at 25°C for 2 hours. Our pilot experiments, as well as previous studies, have shown that polysome profiling is not necessary as long as the digestion with RNase I is complete; rather, this step consumes a large amount of ribosome (especially disome) samples. The following-up computational analyses can help to tell if the RNA fragments being collected are largely protected by ribosomes: whether the fragments are restricted to the coding sequences, whether the fragments are enriched in certain sizes, and whether a 3-nt periodicity exists . Therefore, RNA was directly extracted from the enzyme reaction system with hot phenol and was separated on a 17% (w/v) 7 M urea denaturing polyacrylamide gel in a 0.5×Tris-borate-EDTA (TBE) electrophoresis buffer. RNA fragments with the length of 25-30 nts or 50-80 nts were extracted by gel crushing and further incubated with an RNA gel extraction buffer (300 mM NaOAc pH 5.2, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) overnight. The purified extracted RNA fragments for monosome-seq, disomes-seq, and mRNA-seq were subjected to small RNA library construction for Illumina sequencing (Gnomegen, k02420). The 5′-RNA adaptor contained a 3-nt random sequence at the 3′-end to avoid biased ligation. Monosome and disome footprints were sequenced with single-end 50 and paired-end 100 modes on BGISEQ-500 (BGI Group), respectively. The lengths of RNA fragments protected by single ribosomes peaked at 28-nt, consistent with previous studies.