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| Status |
Public on Aug 10, 2016 |
| Title |
Haloferax_volcanii_1_Fra_TEX_minus |
| Sample type |
SRA |
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| Source name |
Cell culture, Fra, TEX-
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| Organism |
Haloferax volcanii |
| Characteristics |
strain/background: H26
tex: minus
location generated: Frankfurt
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| Growth protocol |
H26 was grown in complex medium under optimal conditions as described previously [PMID: 18493744]. In short, optimal conditions are a temperature of 42oC, a NaCl concentration of 2.2 M, and a good aeration. Cell growth was monitored spectroscopically (OD600) and via counting of the cell density using a Neubauer counting chamber. Cultures were inoculated with exponentially growing pre-cultures and grown to mid-exponential growth phase (4-5 x 10^8 cells ml-1), before they were harvested and used for the RNA-Seq analysis.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cultures were harvested by centrifugation and total RNA was isolated using the RNeasy kit according to the instruction of the manufacturer, including an extensive DNase treatment (Qiagen, Hilden, Germany). The intactness of the RNA was verified by analytical gel electrophoresis, the absence of DNA was verified by comparing PCR reactions with and without a prior cDNA step, and the concentration was determined using a NanoDrop 1000 photometer (Thermo Fisher Scientific).
For Hvolcani_1_Ulm_TEX_plus and Hvolcani_1_Ulm_TEX_minus: The enzymatic treatment with TEX and TAP (tobacco acid pyrophosphatase), RNA adaptor ligation with T4-RNA-ligase, and cDNA synthesis were performed as described [PMID: 20164839]. For the other 4 samples: RNA fractions were sent to vertis (vertis Biotechnologie AG, Martinsried, Germany) for cDNA preparation and high-throughput sequencing. Here, RNA fraction 1 was incubated with TEX to remove the RNA species which carry a 5' mono-phosphate, thus this RNA fraction is enriched in primary transcripts (fraction +TEX). For cDNA synthesis, both RNA fractions were poly(A)-tailed using poly(A) polymerase. This was followed by treatment with TAP to remove the 5' PPP structures. Afterwards, an RNA adapter was ligated to the 5'-monophosphate of the RNAs. First strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified to about 10-20 ng/μl using a high-fidelity DNA polymerase. PCR cycles performed and barcode sequences, which are part of the 3' sequencing adapter. The cDNAs were purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and were analyzed by capillary electrophoresis. For Illumina sequencing, the cDNAs were pooled in approximately equimolar amounts.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
dRNA-Seq
H. volcanii H26 is derived from H. volcanii DS2.
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| Data processing |
FastQ quality trimming using FastX (version 0.0.13) and a cut-off value of 20.
Fastq to fasta conversion using FastX FastX (version 0.0.13).
PolyA removal and size filtering by READemption 0.3.5.
Read alignment using READemption 0.3.5 and segemehl 0.2.0.
Coverage calculation using READemption 0.3.5.
Genome_build: ASM2568v1 (H. volcannii DS2)
Supplementary_files_format_and_content: wiggle
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| Submission date |
Jun 02, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Konrad U. Förstner |
| E-mail(s) |
[email protected]
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| Organization name |
ZB MED - Information Centre for Life Sciences
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| Department |
Information Services
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| Lab |
Förstner Lab
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| Street address |
Gleueler Str. 60
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| City |
Cologne |
| State/province |
North Rhine-Westphalia |
| ZIP/Postal code |
50931 |
| Country |
Germany |
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| Platform ID |
GPL25010 |
| Series (1) |
| GSE82206 |
Genome-wide identification of transcriptional start sites in the haloarchaeon Haloferax volcanii based on differential RNA-Seq (dRNA-Seq) |
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| Relations |
| BioSample |
SAMN05200461 |
| SRA |
SRX1818375 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2186523_Haloferax_volcanii_1_Fra_TEX_minus_forward.wig.gz |
7.3 Mb |
(ftp)(http) |
WIG |
| GSM2186523_Haloferax_volcanii_1_Fra_TEX_minus_reverse.wig.gz |
7.6 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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