SRX12785584 GSM5658041 SRP343289 SRS10731638 GSM5658041 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658041 GSM5658041 GEO Accession GSM5658041 SRX12785586 GSM5658050 SRP343289 SRS10731640 GSM5658050 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658050 GSM5658050 GEO Accession GSM5658050 SRX12785588 GSM5658051 SRP343289 SRS10731642 GSM5658051 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658051 GSM5658051 GEO Accession GSM5658051 SRX12785590 GSM5658052 SRP343289 SRS10731643 GSM5658052 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658052 GSM5658052 GEO Accession GSM5658052 SRX12785592 GSM5658053 SRP343289 SRS10731645 GSM5658053 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658053 GSM5658053 GEO Accession GSM5658053 SRX12785594 GSM5658054 SRP343289 SRS10731647 GSM5658054 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658054 GSM5658054 GEO Accession GSM5658054 SRX12785596 GSM5658055 SRP343289 SRS10731648 GSM5658055 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658055 GSM5658055 GEO Accession GSM5658055 SRX12785598 GSM5658056 SRP343289 SRS10731651 GSM5658056 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658056 GSM5658056 GEO Accession GSM5658056 SRX12785600 GSM5658057 SRP343289 SRS10731653 GSM5658057 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658057 GSM5658057 GEO Accession GSM5658057 SRX12785602 GSM5658058 SRP343289 SRS10731655 GSM5658058 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658058 GSM5658058 GEO Accession GSM5658058 SRX12785604 GSM5658059 SRP343289 SRS10731657 GSM5658059 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658059 GSM5658059 GEO Accession GSM5658059 SRX12785606 GSM5658060 SRP343289 SRS10731659 GSM5658060 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658060 GSM5658060 GEO Accession GSM5658060 SRX12785608 GSM5658061 SRP343289 SRS10731662 GSM5658061 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658061 GSM5658061 GEO Accession GSM5658061 SRX12785610 GSM5658062 SRP343289 SRS10731663 GSM5658062 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658062 GSM5658062 GEO Accession GSM5658062 SRX12785612 GSM5658063 SRP343289 SRS10731666 GSM5658063 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658063 GSM5658063 GEO Accession GSM5658063 SRX12785614 GSM5658064 SRP343289 SRS10731667 GSM5658064 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658064 GSM5658064 GEO Accession GSM5658064 SRX12785616 GSM5658065 SRP343289 SRS10731670 GSM5658065 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658065 GSM5658065 GEO Accession GSM5658065 SRX12785618 GSM5658066 SRP343289 SRS10731671 GSM5658066 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658066 GSM5658066 GEO Accession GSM5658066 SRX12785620 GSM5658067 SRP343289 SRS10731673 GSM5658067 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658067 GSM5658067 GEO Accession GSM5658067 SRX12785622 GSM5658068 SRP343289 SRS10731675 GSM5658068 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658068 GSM5658068 GEO Accession GSM5658068 SRX12785624 GSM5658069 SRP343289 SRS10731677 GSM5658069 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658069 GSM5658069 GEO Accession GSM5658069 SRX12785626 GSM5658070 SRP343289 SRS10731679 GSM5658070 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658070 GSM5658070 GEO Accession GSM5658070 SRX12785628 GSM5658071 SRP343289 SRS10731681 GSM5658071 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658071 GSM5658071 GEO Accession GSM5658071 SRX12785630 GSM5658072 SRP343289 SRS10731683 GSM5658072 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658072 GSM5658072 GEO Accession GSM5658072 SRX12785632 GSM5658073 SRP343289 SRS10731685 GSM5658073 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658073 GSM5658073 GEO Accession GSM5658073 SRX12785634 GSM5658042 SRP343289 SRS10731687 GSM5658042 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658042 GSM5658042 GEO Accession GSM5658042 SRX12785636 GSM5658043 SRP343289 SRS10731689 GSM5658043 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658043 GSM5658043 GEO Accession GSM5658043 SRX12785638 GSM5658044 SRP343289 SRS10731691 GSM5658044 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658044 GSM5658044 GEO Accession GSM5658044 SRX12785640 GSM5658045 SRP343289 SRS10731693 GSM5658045 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658045 GSM5658045 GEO Accession GSM5658045 SRX12785642 GSM5658046 SRP343289 SRS10731696 GSM5658046 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658046 GSM5658046 GEO Accession GSM5658046 SRX12785644 GSM5658047 SRP343289 SRS10731697 GSM5658047 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658047 GSM5658047 GEO Accession GSM5658047 SRX12785646 GSM5658048 SRP343289 SRS10731698 GSM5658048 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658048 GSM5658048 GEO Accession GSM5658048 SRX12785648 GSM5658049 SRP343289 SRS10731701 GSM5658049 RNA-Seq TRANSCRIPTOMIC cDNA 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. MinION gds 305658049 GSM5658049 GEO Accession GSM5658049