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| Status |
Public on Apr 23, 2021 |
| Title |
Yeast_rRNA_PolysomeF3.rep1 |
| Sample type |
SRA |
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| Source name |
Yeast_rRNA_PolysomeF3
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: BY-4741
genotype: WT
sucrose gradient fraction: Fraction 3
rna fraction: Ribosomal RNA
stress: Normal
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| Growth protocol |
Saccharomyces cerevisiae strains were grown at 30 ˚C in standard YPD medium (1% yeast extract, 2% Bacto Peptone and 2% dextrose). The deletion strains snR3Δ, snR34Δ and snR36Δ were generated on the background of the BY4741 strain by replacing the genomic snoRNA sequence with a kanMX4 cassette as detailed in Parker et al. [57]. For the analysis of rRNAs modifications across polysomal fractions, yeast BY4741 starter cultures were grown in 6 mL YPD medium at 30 ºC with shaking (250 rpm) overnight. 100 mL of fresh YPD medium was inoculated with 10 µL of the stationary culture in a 250 mL erlenmeyer flask, in biological duplicates. Cells were incubated at 30 ºC with shaking (250 rpm) until the cultures reached mid-exponential growth phase (O.D660.~ 0.5-1.0). Yeast cells were then treated with 1 mM H202 or left without treatment (control) for 30 min. 1 mL of cycloheximide stock solution (10 mg/mL) was added to each culture. After 5 min incubation, cells were then quickly transferred into 50 mL pre-chilled falcon tubes, and centrifuged for 5 min at 3,000 g in a 4 ºC pre-chilled centrifuge. Supernatant was discarded, and cells were flash frozen.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For the samples 1-8 and 17-18-21-22:Saccharomyces cerevisiae BY4741 cells (strains: snR3Δ, snR34Δ snR36Δ, and WT) were harvested via centrifugation at 3000 RPM for 1 minute, followed by two washes with water. RNA was purified from pelleted cells using a MasterPure Yeast RNA extraction kit (Lucigen, MPY03100), according to manufacturer’s instructions., For the rest (Fractions): Yeast pellets from 100 mL cultures were washed with 6 mL of ice-cold Polysome Extraction Buffer (PEB), which contained 20 mM Tris-HCl pH 7.4, 100 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mg/mL cycloheximide and 100 U/mL RNAse inhibitors (RNaseOUT, Invitrogen, #18080051). Cells were centrifuged for 5 min at 3,000 g at 4ºC. Washing was repeated by adding 6 mL of ice-cold PEB, followed by centrifugation. Cells were then resuspended in 700 uL of ice-cold PEB, and transferred into pre-chilled 2 mL Eppendorf tubes containing 450 uL of pre-chilled RNAse-free 425-600 μm diameter glass beads (Sigma G8772). Cells were lysed by vortexing for 5 min at 4 ºC, followed by centrifugation at maximum speed at bench centrifuge for 5 min at 4ºC. 10% of the supernatant was aliquoted into Trizol for total RNA isolation, and kept at -80ºC. The remaining volume, corresponding approximately to 8 x 10e8 cells, was subsequently loaded onto the sucrose gradient. Linear sucrose gradients of 10-50% were prepared using the Gradient Station (BioComp). Briefly, SW41 centrifugation tubes (Beckman, Ultra-ClearTM) were filled with Gradient Solution 1 (GS1), which consisted of 20 mM Tris-HCl pH 7.4, 100 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mg/mL cycloheximide and 10% w/v RNAse-free sucrose. The tube was then filled with 6.3 mL of Gradient Solution 2 (GS2) layered at the bottom of the tube, which consisted of 20 mM Tris-HCl pH 7.4, 100 mM KCl, 10 mM MgCl2, 0.5 mM DTT, 0.1 mg/mL cycloheximide and 50% w/v RNAse-free sucrose. The linear gradient was formed using the tilted methodology, with the Gradient Station Maker (Biocomp). Once the gradients were formed, 400 µL of each lysate was carefully loaded on top of the gradients, and tubes were balanced in pairs, placed into pre-chilled SW41 buckets and centrifuged for 150 min, at 35,000 RPM. Gradients were then immediately fractionated using the Gradient Station, and 20 x 500 uL fractions were collected in 1.5 mL Eppendorf tubes, while absorbance was monitored at 260 nm continuously. Fractions were combined in the following way: the free rRNA (F1), the unassembled subunits (F2), the lowly-translating monosomes (F3) and the highly-translating polysomes (F4). The pooled fractions were then concentrated using Amicon-Ultra 100K columns (Millipore), and washed two times with PEB. The final volume was brought down to 200 µL, and RNA was extracted using TRIzol reagent.
Yeast total RNA was first treated with T4 Polynucleotide Kinase (PNK) (NEB M0201S) in order to remove possible phosphorylated 3’ends before polyA tailing. Briefly, we mixed 1 ul PNK enzyme with rRNAs in a 50 ul reaction with T4 PNK Buffer (10X) and incubated at 37°C for 30 minutes. The products were purified using 1.8X Agencourt RNAClean XP beads (Fisher Scientific-NC0068576), washing with 70% freshly prepared ethanol. Dephosphorylated rRNAs were then polyadenylated using E.coli Poly(A) Polymerase, following the commercial protocol. Four different direct RNA libraries were barcoded according to the recent protocol that our lab has recently published [58]. Custom RT adaptors (IDT) were annealed using following conditions: custom Oligo A and B (Table S4) were mixed in annealing buffer (0.01M Tris-Cl pH7.5, 0.05M NaCl) to the final concentration of 1.4 uM each in a total volume of 75 ul. The mixture was incubated at 94 °C for 5 minutes and slowly cooled down (-0.1 °C/s) to room temperature. RNA library for direct RNA Sequencing (SQK-RNA002) was prepared following the ONT Direct RNA Sequencing protocol version DRS_9080_v2_revI_14Aug2019 with half reaction for each library until the RNA Adapter (RMX) ligation step. Per reaction (half), 250 ng total of polyA tailed yeast rRNAs were ligated to pre-annealed custom RT adaptors (IDT) [58] (Table S4) using concentrated T4 DNA Ligase (NEB-M0202T), and was reverse transcribed using Maxima H Minus RT (Thermo Scientific, EP0752), without heat inactivation step. The products were purified using 1.8X Agencourt RNAClean XP beads (Fisher Scientific-NC0068576), washing with 70% freshly prepared ethanol. 50 ng of reverse transcribed RNA from each reaction was pooled and RMX adapter, composed of sequencing adapters with motor protein, was ligated onto the RNA:DNA hybrid and the mix was purified using 1X Agencourt RNAClean XP beads, washing with Wash Buffer (WSB) twice. The sample was then eluted in Elution Buffer (EB) and mixed with RNA Running Buffer (RRB) prior to loading onto a primed R9.4.1 flow cell, and ran on a MinION sequencer with MinKNOW acquisition software version v.3.5.5..
Nanopore Direct RNA Sequencing
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
MinION |
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| Description |
Sample 11
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| Data processing |
Basecalling with Guppy 3.0.3
Demultiplexing with Deeplexicon
Alignment with Graphmap Default settings
Epinano performed to extract base-called features per position/per 5mer
Nanopolish performed to extract aligned current intensities and subsequently, mean values per position were calculated using a python script
Genome_build: Saccharomyces cerevisiae S288c ribosomal RNA sequences
Supplementary_files_format_and_content: Epinano outputs contain base-called feautures (base quality, mismatch, insertion, deletion frequencies) for each position. Nanopolish outputs contain current intensity mean values for each position
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| Submission date |
Apr 13, 2020 |
| Last update date |
Apr 23, 2021 |
| Contact name |
Eva Maria Novoa |
| E-mail(s) |
evamaria.novoa@gmail.com
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| Phone |
+34933160260
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| Organization name |
Center for Genomic Regulation
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| Lab |
Epitranscriptomics and RNA Dynamics
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| Street address |
Dr. Aiguader 88
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| City |
Barcelona |
| ZIP/Postal code |
08003 |
| Country |
Spain |
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| Platform ID |
GPL25739 |
| Series (1) |
| GSE148603 |
Quantitative profiling of native RNA modifications and their dynamics using nanopore sequencing |
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| Relations |
| BioSample |
SAMN14593329 |
| SRA |
SRX8111380 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4475016_f3_rep1_EpiNano_features.csv.gz |
204.7 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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