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Sample GSM4034885

Query DataSets for GSM4034885

Status

Public on Jul 23, 2020

Title

Ssol_mock

Sample type

SRA

Source name

archaeal cells

Organism

Saccharolobus solfataricus

Characteristics

genetic background: WT
treatment: mock
growth condition: 85 degrees

Treatment protocol

HEK-293T cells transfected with 3xFLAG-tagged NAT10 and myc-tagged THUMPD1 using FuGENE® 6 transfection reagent (Promega, #E2691) 24 hours prior to harvesting.

Growth protocol

T. kodakarensis and T. sp AM4 strains – TS559 can derivatives thereof – were grown as previously described (Santangelo et al. 2007; Hileman and Santangelo 2012; Gehring et al. 2017) in artificial sea water (ASW) medium supplemented with vitamins and trace minerals. Pyrococcus furiosus strain COM1 was cultured at 75-95˚C in an artificial sea water based medium supplemented with cellobiose, maltose, yeast extract, S˚, trace minerals, cysteine and sodium tungstate as previously described (Lipscomb et al, APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2011, p. 2232–2238).
HEK-293T and HeLa (WT and Nat10 depleted) cells were grown in DMEM media.
Saccharomyces cerevisiae strains were grown at 30°C in standard YEP medium (1% yeast extract, 2% Bacto Peptone) supplemented with 2% galactose (YPG), or 2% dextrose (YPD).

Extracted molecule

total RNA

Extraction 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

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NextSeq 500

Data processing

Library strategy: ac4C-seq
alignment using STAR (V. 2.5.3a)
base-calling using JACUSA v2.0.0 under default parameters
calculation of misincorporation rates per base
Genome_build: ASM996v1 for Thermococcus kodakarensis, ASM27560v1 for Pyrococcus furiosus, ASM15120v2 was used for Thermococcus sp. AM4, ASM700v1 for Sulfolobus solfataricus and ASM9166v1 for Methanocaldococcus jannaschii. For human GRCh37/hg19 with UCSC Genes annotations. For Saccharomyces cerevisiae samples the sacCer3 assembly.
Supplementary_files_format_and_content: SasChen_TableS2.xlsx an excel file detailing ac4C sites identified in each organism including the level of modification of each site.

Submission date

Aug 14, 2019

Last update date

Jul 23, 2020

Contact name

Aldema Sas-Chen

E-mail(s)

aldema.sas@gmail.com

Organization name

Tel Aviv University

Department

Shmunis School of Biomedicine and Cancer Research