|
| Status |
Public on Nov 24, 2020 |
| Title |
Sso-RNAseq rep3 |
| Sample type |
SRA |
| |
|
| Source name |
S. solfataricus P2
|
| Organism |
Saccharolobus solfataricus |
| Characteristics |
density: OD600 of 0.8
|
| Treatment protocol |
Individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) analyses were performed as described in König et al. (2010) (PMID: 20601959), with the following alteration that were necessary for the application in S. solfataricus. Cell pellets were washed with PBS, and 0.8 g were resuspended in 40 ml PBS at 4°C, and spread on a large petri dish swimming on an ice-water bath. The cell suspension was crosslinked four times at 254 nm, 300 mJ/cm², cells were mixed in between. Crosslinked cells were harvested as before and stored at -80°C.
|
| Growth protocol |
S. solfataricus P2 was grown as previously described in a 10 l bioreactor (Evguenieva-Hackenberg et al., 2002) (PMID: 12359717). Cells were cultivated until an OD600 of 0.8 and harvested by centrifugation at 6,000 g and 4 °C for 10 min.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA samples were first fragmented using ultrasound (4 pulses of 30 sec at 4°C). Each sample was used for three independent cDNA synthesis. First, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3’ adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis.
The total RNA samples were first fragmented using ultrasound (4 pulses of 30 sec at 4°C). Each sample was used for three independent cDNA synthesis. First, an oligonucleotide adapter was ligated to the 3' end of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3’ adapter as primer. The first-strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
alignment: bowtie2 (v2.3.4) –sensitive
counts: featureCounts
Genome_build: ensembl Sulfolobus_solfataricus_p2.ASM700v1:
Supplementary_files_format_and_content: counts
|
| |
|
| Submission date |
Apr 22, 2020 |
| Last update date |
Nov 24, 2020 |
| Contact name |
Gabriele Klug |
| E-mail(s) |
[email protected]
|
| Organization name |
Justus-Liebig University Gießen
|
| Department |
Molecular- and Microbiology
|
| Street address |
Heinrich-Buff-Ring 26-32
|
| City |
Gießen |
| ZIP/Postal code |
35392 |
| Country |
Germany |
| |
|
| Platform ID |
GPL27060 |
| Series (2) |
| GSE149142 |
iCLIP analysis of RNA substrates of the archaeal exosome (RNA-Seq) |
| GSE149143 |
iCLIP analysis of RNA substrates of the archaeal exosome |
|
| Relations |
| BioSample |
SAMN14670179 |
| SRA |
SRX8156865 |
