GSM5658055: 20210618_R941_Mix; Saccharomyces cerevisiae; RNA-Seq
RNA was extracted from approximately five total OD600 of cells (usually 10 ml culture at OD600 = 0.5 for mid log cells, 0.5 ml of stationary cells at OD600 = 10) using a hot phenol protocol 1 described in (Ares 2012). Direct RNA sequencing libraries were constructed using the SQK-RNA002 (Oxford Nanopore Technologies) kit following the manufacturer's protocol with the following modifications.  Briefly, 750 ng of total yeast RNA was used as input material.  To facilitate ligation of sequencing adapters to endogenous yeast 18S and 25S rRNA, 1 ul of 10 pmol/ul custom oligonucleotide duplexes complementary to the 3' ends of 18S and 25S rRNA and the 5' end of the ONT RMX sequencing adapter were used instead of the kit provided RTA adapter (Supplemental Table S5).  To create duplexes, 100 pmol of either 18S or 25S splint oligo was incubated with 100 pmol of sequencing adapter and nuclease free H20 in a total volume of 10 ul.  Reactions were heated to 95 °C for 2 minutes and gradually cooled at 65 °C for 10 minutes, 48 °C for 10 minutes, room temperature for 10 minutes and then placed on ice. Annealed oligonucleotide duplexes targeting 18S and 25S rRNAs were then pooled in equimolar ratio and 1 ul of the pool was used for sequencing library preparation. In the case of T7 rRNA sequencing libraries, T7-18S splint and T7-25S splint oligos were used to capture the 3' end generated by HindIII digestion and run-off transcription.  To enhance ligation efficiency during library preparation, the first and second ligation steps were increased from 10 minutes to 15 minutes and performed at room temperature. Reverse transcription was omitted. Sequencing-adapted libraries were eluted in 21 ul of elution buffer.