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| Status |
Public on Dec 03, 2021 |
| Title |
20210729_R941_dbr1d_YPD |
| Sample type |
SRA |
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| Source name |
20210729_R941_dbr1d_YPD
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY4741 dbr1<delta>::KanMX4
strain information: MATa; his3<delta>1; leu2<delta>0; met15<delta>0; ura3<delta>0; dbr1<delta>::KanMX4
genotype/treatment: Dbr1 KO
stress: None
molecule subtype: ribosomal RNA
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| Treatment protocol |
Unless indicated, all other strains were grown in YEPD at 30 °C to mid-log phase. Cells exposed to various environmental conditions were treated as follows: 1% KOAc (1 hr, 30 °C), cycloheximide (1 ug/ml for 1 hour), rapamycin (200 ng/ml for 1 or 5 hours), and pladienolide B (5 uM for 1 hour). Stationary phase cells were grown to an OD600 = 10. Strains carrying prp16-302 (Madhani and Guthrie 1994) and prp43 Q423N (Leeds et al. 2006) mutations, and wild type (Schattner et al. 2004) were grown to mid log phase at 30 °C and shifted to 18 °C for 1 hour by addition of an equal volume of 6 °C YEPD.
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| Growth protocol |
Yeast strains GAL-NOP58 and GAL-CBF5 are described in (Lafontaine and Tollervey 1999)(Lafontaine et al. 1998). Cells were grown at 30 °C in YEPgal liquid medium (2% galactose, 2% peptone, 1% yeast extract) or shifted to liquid YEPD (2% dextrose, 2% peptone, 1% yeast extract) to mid-log phase (OD600 = 0.25-0.5) for 16 hours to repress expression of Nop58 or Cbf5. Cells were harvested by centrifugation and RNA was isolated. The spp382-1 strain is described in (Pandit et al. 2006). The strains deleted for the SNR80 (YWD448a), SNR83 (YWD451a) or SNR87 (YWD452a) genes are described in (Schattner et al. 2004). Yeast strains deleted for the SNR4 and SNR45 genes are described in (Parker et al. 2018).
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| Extracted molecule |
total RNA |
| Extraction protocol |
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.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
MinION |
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| Data processing |
Basecalling with Guppy 3.1.5+781ed57
split fast5's with multi_to_single_fast5
index with nanopolish
align to yeast 18S and 25S rRNA reference
signalAlign produced posterior probabilities of events aligned to reference kmers
embed_fast5 aggregated posterior probabilites to produce probability of modification
Genome_build: Saccharomyces cerevisiae S288c ribosomal RNA sequences
Supplementary_files_format_and_content: embed_fast5 outputs contain modification calls (read_id,contig,reference_index,strand,variants,prob1,prob2,prob3,prob4) for each modification site for each read. Prob1 is probability of being unmodified and prob2 is modified. Prob3 and prob4 are not used.
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| Submission date |
Oct 26, 2021 |
| Last update date |
Dec 04, 2021 |
| Contact name |
Manuel Ares |
| E-mail(s) |
ares@ucsc.edu
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| Organization name |
University of California, Santa Cruz
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| Street address |
1156 High St
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| City |
Santa Cruz |
| State/province |
CA |
| ZIP/Postal code |
95064 |
| Country |
USA |
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| Platform ID |
GPL25739 |
| Series (1) |
| GSE186634 |
Concerted modification of nucleotides at functional centers of the ribosome revealed by single-molecule RNA modification profiling |
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| Relations |
| BioSample |
SAMN22589443 |
| SRA |
SRX12785610 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5658062_20210729_R941_dbr1d_YPD.csv.gz |
2.2 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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