GSM4034885: Ssol_mock; Saccharolobus solfataricus; OTHER - SRA

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SRX6714214: GSM4034885: Ssol_mock; Saccharolobus solfataricus; OTHER
1 ILLUMINA (NextSeq 500) run: 62.6M spots, 5.3G bases, 2.1Gb downloads

Submitted by: NCBI (GEO)

Study: Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping

PRJNA560218 • SRP218339 • All experiments • All runs

show Abstracthide Abstract

N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification, present on tRNA, rRNA and recently investigated in eukaryotic mRNA. We report ac4C-seq, a chemical genomic method for single-nucleotide resolution, transcriptome-wide quantitative mapping of ac4C. While we did not find detectable ac4C sites in human and yeast mRNAs, ac4C was induced via ectopic overexpression of eukaryotic acetyltransferase complexes, invariably at a conserved sequence motif. In contrast, cross-evolutionary profiling reveals unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, ncRNA and mRNA from hyperthermophilic archaea. Ac4C is dramatically induced in response to temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Cryo-EM visualization of WT and acetyltransferase-deficient archaeal ribosomes furnishes structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for unravelling this modification's role in biology and disease. Overall design: polyA-enriched RNA from HEK-293T WT (single sample) or Nat10+Thumpd1 overexpressing (2 replicates) cells under NaCNBH3, deacetylation or mock treatment. Total RNA from HeLa WT or Nat10 depleted cells (3 replicates each) under NaCNBH3, deacetylation or mock treatment. polyA-enriched RNA from HeLa WT (3 replicates) or Nat10 depleted (2 replicates) cells under NaCNBH3 or mock treatment. Total RNA from M. jannaschiae under NaCNBH3, deacetylation or mock treatment. Total RNA from S. solfataricus under NaCNBH3, deacetylation or mock treatment. Total RNA from T. sp. AM4 under NaCNBH3 or mock treatment, grown in 65, 75 and 85 degrees celsius. Total RNA from P. furiosus under NaCNBH3 or mock treatment, grown in 75, 85 and 95 degrees celsius. Total RNA from T. kodakarensis (T.kod) under NaCNBH3 or mock treatment, grown in 55 (1 sample), 65 (2 samples), 75 (2 samples), 85 (4 samples) and 95 (1 sample) degrees celsius. One of the replicates of 85 degree of T.kod underwent also deacetylation. Total RNA from T. kod strains deleted for TK0754 or TK2097 grown at 85 degrees and treated with NaCMBH3 or mock (in duplicates). T.kod purified ribosomes treated with NaCNBH3. rRNA-depleted RNA from WT T.kod grown at 85 and 95 degrees were treatedd with NaCNBH3 or NaCNBH3 and mock, respectively.

Sample: Ssol_mock

SAMN12572382 • SRS5267194 • All experiments • All runs

Organism: Saccharolobus solfataricus

Library:

Instrument: NextSeq 500

Strategy: OTHER

Source: TRANSCRIPTOMIC

Selection: other

Layout: PAIRED

Construction protocol: Total RNA was isolated from T. kodakarensis using TRIZOL according to manufacturer's protocol. For the indicated samples of T. kodakarensis ,rRNAs were depleted according to Morlan et al.(Morlan et al. 2012) using reagents provided in the NEBNext® rRNA Depletion Kit (NEB #E6310). For indicated samples purifications of T. kodakarensis ribosomes of the wild-type and TkNat10 knockout were conducted similar to previously documented procedures (Matzov et al. 2017). Total RNA from Human Cells was extracted using TRIZOL according to manufacturer's protocol. For the indicated samples Poly(A) RNA from yeast and human total RNA was isolated by two rounds of purification using the GenElute mRNA miniprep kit (Sigma) according to manufacturer's protocol. 500ug total RNA was used per purification column. Typical yield after two rounds of isolation was 1.2%. Total RNA was isolated from Yeast using hot acidic phenol. Briefly, frozen yeast (S. cerevisiae) pellet suspended in 1.0 mL AES buffer (50mM NaOAc, 10mM EDTA ph 8.0, 1% SDS) per 0.5mL pellet volume. To suspended pellet, 1.0 mL Acid buffered Phenol per mL of AES buffer used was added. Sample was mixed by vortexing and incubated in a 65˚C water bath for 30 min, vortexing every 2 minutes to mix. Samples were put on ice for 10 minutes and 1.0 mL Chloroform:isoamyl alcohol (24:1) was added for each 1.0 mL phenol used. Sample was vortexed to mix and centrifuged 5000 rcf for 15 minutes. Aqueous layer (top) was transferred to a clean tube and extracted 3X with an equal volume of acid buffered phenol:chlorofom:isoamyl alcohol (24:23:1). After each extraction sample was centrifuged at 5000 rcf for 10 minutes and aqueous layer transferred to a new tube. A final extraction with chloroform:isoamyl alcohol was carried out to remove residual phenol. Aqueous layer was transferred to a clean tube and RNA was precipitated by the addition of an equal volume of 100% isopropanol and 1/9th volume of 3M Sodium Acetate. Samples were incubated -20˚C for 30 minutes and centrifuged 12000 rcf at 4˚C for 15 minutes. Supernatant was decanted and the pellet washed with 4 mL ice fold 70% ethanol. RNA pellet was resuspended by briefly heating at 50 ˚C in 1.0 mL 1X TE buffer pH 8.0. Samples were quantified by UV absorbance and stored at -80˚C. Typical extractions were carried out with 1.0 mL volume cell pellets and yielded 20mg of total RNA Fragmentation, 3' adapter ligation, cDNA synthesis, second adapter ligation and enrichment

Links:

NCBI link: NCBI Entrez (gds)

Runs: 1 run, 62.6M spots, 5.3G bases, 2.1Gb

Run	# of Spots	# of Bases	Size	Published
SRR9966726	62,642,615	5.3G	2.1Gb	2020-07-23

ID:: 8846191

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