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| Status |
Public on Oct 26, 2021 |
| Title |
pombe_dus3_R_II |
| Sample type |
SRA |
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| Source name |
S. pombe cells
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: deltadus3
replicate: II
treatment: R+
percentage of d in spike-in: 0
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| Growth protocol |
50 mL of S. pombe grown in YES at 32°C (OD595nm ~ 0.5) or E. coli grown in LB at 37°C (OD595nm ~ 0.6) or HCT116 human cells were harvested by centrifugation and washed in DEPC water.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The cell pellets were resuspended in 750μL of TES buffer (10mM Tris pH 7.5, 10mM EDTA pH 8, 0.5% SDS) and the total RNA was isolated by hot phenol extraction. Rhodamine labeling was performed as described in our manuscript. Briefly, 30μg of total RNA were resuspended in 1% sodium borohydride for 60min at 25°C. Mock samples (R-) were treated similarly but without sodium borohydride. The reduced RNA was then precipitated and the dried pellet was resuspended in 0.1M sodium formate pH 4. Rhodamine labeling was performed for 90min at 37°C in dark with 2.2mM of rhodamine chlorine. Labeled RNA was purified by phenol extraction.
A synthetic dihydrouridilated RNA spike-in was added to the samples (1:1000) before ribodepletion of 5 μg of the RNA samples. Zinc-mediated fragmentation was performed at 70°C for 15min with 90-125 ng of ribodepleted RNA. Simultaneous DNA digestion and RNA extremities dephosphorylation were performed at 37°C for 30min with 2U of TURBO DNase and 3U of FastAP Thermosensitive Alkaline Phosphatase. 3'-end ligation of the RNA adapter was performed for 90min at 25°C with 39U of T4 RNA ligase. After ligation, 10pmol of DNA primer were added, the sample was denatured for 2min at 70°C and the reverse transcription was performed with Superscript III. The excess of DNA primer was subsequently digested with 4min incubation with 2μL of ExoSAP-IT. The 3'-end ligation of the cDNA with DNA adapter was performed overnight at 25°C with 48U of T4 RNA ligase. Nucleic acid clean-ups were performed between each step by using MyOne SILANE magnetic beads. 13 cycles od PCR amplification was performed in a 50μL-reaction by mixing cDNA, 1μM of barcoded PCR primer, 1μM of universal PCR primer and 25μL of NEBNext High- Fidelity 2X PCR Master Mix (NEB M0541). Finally, two successive rounds of DNA polymer size selection were performed by mixing PCR products with 1.2 and 1.1 volume of AMPure XP beads. The DNA library was quantified, examined on a DNA microchip and sequenced on Illumina HiSeq 2500 or Novaseq.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Library strategy: Rho-seq
Reads were quality- and adaptor-trimmed using trimmomatic 0.36 with options PE ILLUMINACLIP:TruSeq2-PE.fa:2:30:10 HEADCROP:1 SLIDINGWINDOW:4:15 MINLEN:20
The filtered reads were mapped on the spike-in sequence with bowtie2 (options --no-discordant --no-mixed --phred33 -p 4 --un-conc) then on the genome using also bowtie2 for the E.coli libraries or tophat2 for the S. pombe libraries (options--mate-std-dev 150 -N 2 --read-edit-dist 2 --min-intron-length 5 --max-intron-length 3000 --no-discordant --no-coverage-search -p 4 --library-type fr-firststrand).
Properly paired mapped reads were split by strand. Then bedtools (options genomecov -ibam -d -5) was used to calculate the number of RT termination events for each strand-specific position as the number of 5'end of R2 reads that map at this position. In parallel, the paired reads were computationally extended/merged into fragments that covered the genome from the left end of the left-most read to the right end of the right-most reads with the notable exeption that the fragments were not extended in identified introns. Those spliced fragments where then used to compute a fragment coverage reflecting the number of time a reverse transcriptase passed through a specific position. Then the ratio between the number of RT termination events and fragment coverage at each position (the D-ratio) was calculated.
After independent pre-filtering of the positions unlikely to have significant differences in D-ratio accross conditions (see manuscrit), the number of RT termination events out of the number of RT readthrough were modelised in generalized linear model of the binomial family (function glm in R) according to the effect of the treatment (R+ vs R-), the effect of the strain (wild-type vs delta4dus), and their interaction. The positions were the interaction between the wild-type and the delta4dus lead to a statistically significant (FDR<0.1) increase of the proportion of RT termination events were retained as putative dihydrouridilation sites.
Genome_build: ASM294v2.26 (pombe), ASM584v2.38 (coli), hg38 (human)
Supplementary_files_format_and_content: Rho-seq_supplementary.xlsx is a spreadsheet describing the dihydrouridilated sites (D-sites) uncovered in our study. The first three sheets describe the D-sites found respectively on the S. pombe, the E. coli and human transcriptome, while the third sheet contains a legend explaining the column names in the first three sheets.
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| Submission date |
Oct 08, 2021 |
| Last update date |
Oct 26, 2021 |
| Contact name |
Carlo Yague-Sanz |
| E-mail(s) |
[email protected]
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| Organization name |
Namur University
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| Department |
Medecine
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| Lab |
GEMO lab
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| Street address |
61 rue de Bruxelles
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| City |
Namur |
| ZIP/Postal code |
5000 |
| Country |
Belgium |
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| Platform ID |
GPL28961 |
| Series (2) |
| GSE145685 |
Transcription-wide distribution of dihydrouridine (D) into mRNAs reveals its requirement for meiotic chromosome segregation. [Rho-Seq] |
| GSE145686 |
Transcription-wide distribution of dihydrouridine (D) into mRNAs reveals its requirement for meiotic chromosome segregation. |
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| Relations |
| BioSample |
SAMN22148619 |
| SRA |
SRX12528249 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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