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Sample GSM1348917

Query DataSets for GSM1348917

Status

Public on Jul 25, 2014

Title

Af_0min+O2-A1

Sample type

SRA

Source name

hyphae

Organism

Aspergillus fumigatus

Characteristics

strain: CEA 10
cell type: none
treatment protocol: timepoint 0 min, 100 % oxygen saturation
cultivation time (minutes): 0

Treatment protocol

Cultures were aerated with a continuous flow of air to maintain O2 saturation at 100% (≈260 µmol l-1) at an initial gas flow rate of 0.65 l min-1. After an initial growth phase of 16-18 h (timepoint 0 min) cultures were rapidly shifted to microaerobic conditions by switching to a preadjusted ratio of N2 and air which lowered the initial O2 concentration down to 5% and samples were taken after 15 and 30 min (timepoint 15 and 30 min, respectively). After 60 min, full aeration restored 100% O2 saturation and a last sample was taken after 15 min (timepoint 75 min).

Growth protocol

To analyze the low oxygen response, the fungus was grown in an O2-controlled fermenter (Biostat B-DCU-5l, Braun, Melsungen, Germany), essentially as described earlier (Barker et al., 2012). Briefly, conidia were inoculated in AMM with 1% [w/v] of Glucose as the sole source of carbon at a final concentration of 105 ml-1 in a total culture volume of 4 l at 37°C and a constant stirring rate of 250 rpm. A pO2 electrode (InPro6800/12/320, Mettler-Toledo, Steinbach, Germany) was used to monitor the concentration of dissolved O2.

Extracted molecule

total RNA

Extraction protocol

Culture aliquots of 200 ml were taken before and at indicated time points throughout the shift. The mycelium was rapidly separated from the medium by filtration, immediately shock frozen in liquid N2 and stored at -80°C until RNA isolation. A phenol-chloroform based method was used to extract total RNA from mycelia. First, 100-mg-aliquots of the deeply frozen mycelium of A. fumigatus were mechanically disintegrated in the presence of 1 ml of TriSureTM (Bioline GmbH, Luckenwalde, Germany) using the FastPrep®24 instrument (MP Biomedicals, Illkirch, France). Following the addition of chloroform and centrifugation, RNA was precipitated from the aqueous phase and solubilized in ultrapure H2O.RNA was e additionally digested with DNaseI and column purified using the RNeasy spin columns (Qiagen, Hilden, Germany) to remove DNA and phenol contaminations, respectively. Concentration and integrity of the RNA were checked by Nanodrop measurements and agarose gel electrophoresis.
tagged random primed cDNA libraries were prepared from fragmented polyA-RNA using standard illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Data processing

FASTQ files were received from GATC, Konstanz, Germany
Sequenced reads were trimmed for low-quality regions (btrim windowsize 15 average 20)
Mapping of reads using TopHat (v2.0.6; -G GFF from CADRE (Central Aspergillus Data REpository); -maxintronlength 10000)
Read counting using htseq-count (-mode union, -s no, -type CDS)
Calculation of RPKM values using raw counts, sum of totally mapped reads and sum of CDS length using CADRE GFF file
Genome_build: Aspergillus fumigatus CEA10 (CADRE)
Supplementary_files_format_and_content: xls

Submission date

Mar 17, 2014

Last update date

May 15, 2019

Contact name

Steffen Priebe

E-mail(s)

[email protected]

Organization name

Hans-Knöll-Institute (HKI)

Department

Systems Biology / Bioinformatics