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| Status |
Public on Nov 03, 2017 |
| Title |
Esi_ex_3 |
| Sample type |
SRA |
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| Source name |
DSMZ 17290
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| Organism |
Exiguobacterium artemiae |
| Characteristics |
strain: 255-15
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| Treatment protocol |
cells were harvested by centrifugation at 2800 x g and processes further directly.
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| Growth protocol |
growth temperature: 20°C
For a preculture cells were grown in media suggested by DSMZ at the optimal temperature over night. Main cultures were inoculated at OD600 = 0.2 and grown to mid log phase. Half of the culture was harvested as the sample for exponential growth and the other half cultured over night until 24h of incubation time was reached. The remaining culture was harvested as the stationary samples. Each culture was run in triplicates.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Immediately after harvesting cells were treated with TriZOL to isolated total RNA as recommended by the manufacturer. Cell disruption was fascilitated by using FastPrep24 (6 m/s, 30 sec). To precipiated larger RNA species, samples were subjected to high salt precipitation using 5 M LiCl and the supernatant was desalted via G-25 spin filtration (PD-10; GE). RNA was ethanol precipitated and subjected to RiboZero treatment (Samples 3, 6, 9, 12, 15, 18, 23) or processed without further treatment.
A 3'-blocked oligonucleotide was adenylated (NEB #E2610) and ligated to the isolated RNA with a truncated T4-RNA-Ligase 2 (NEB #M0242). Ligation products were reverse transcribed (RevertAid; Thermo Scientific) using a 32P 5'-labeled primer and resulting cDNA was isolated from 10% denaturing gel. A pre-adenylated 3'-blocked oligonucleotide was ligated to isolated cDNA using a 5’-App-DNA/RNA-Ligase (NEB #M0319) at conditions given for ssDNA ligation. Ligation products were amplified by PCR in a 100 µL reaction for 15-16 cycles and isolated from MethylBlue stained gel. For each sample a indexed was introduced during PCR and one lane on MiSeq was used per replicate. The third sample from B. subtilis from stationary phase yielded a not sufficient DNA amount and did not generate usable sequence results.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Description |
tRNA
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| Data processing |
Genomes of Pha, Esi, Gst, and Bsu were scanned for tRNAs using tRNAscan-SE 1.3.1.
Sequenced reads were trimmed for adaptor sequences using cutadapt 1.11 and mapped to the corresponding set of genomic tRNAs counting all mapped as well as the different 3’-ends of the transcripts.
Genome_build: Bsu (NC_000964.3), Esi (NC_010556.1), Gst (NZ_CM002692.1), and Pha (NZ_CP016537.2)
Supplementary_files_format_and_content: Data files contain tRNA gene annotations and corresponding total read counts as well as read counts for different 3’-ends of tRNA transcripts.
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| Submission date |
May 12, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Felix Georg Maria Ernst |
| Organization name |
University of Leipzig
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| Department |
Faculty of Bioscience
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| Lab |
Molcular Biology
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| Street address |
Bruederstrasse 34
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| City |
Leipzig |
| ZIP/Postal code |
04103 |
| Country |
Germany |
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| Platform ID |
GPL23471 |
| Series (1) |
| GSE98874 |
Cold adaptation of tRNA nucleotidyltransferases: a tradeoff in activity, stability and fidelity |
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| Relations |
| BioSample |
SAMN06958561 |
| SRA |
SRX2804658 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2617014_Esi-ex-3.csv.gz |
1.7 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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