title: Wheat bran, replicate 1
organism: Malbranchea cinnamomea
description: Protocols: The fungus used in the present study, was isolated from compost at the Cau Dien municipal waste treatment factory in Hanoi, Vietnam (geographical location 21°01'01.7\N, 105°45'09.2\E, 11 m [longitude, latitude, altitude]), and was grown on potato dextrose agar (PDA) at 50°C. DNA was extracted for identification of the ITS region with primers ITS1 and ITS4. 2 x 200 mL of basal medium containing glucose (20 g L-1) as the carbon source were prepared. The basal medium had the following composition: 4 g L-1 KH2PO4, 13.6 g L-1 (NH4)2SO4, 0.8 g L-1 CaCl2-2H2O, 0.6 g L-1 MgSO4-7H2O, 6 g L-1 Bacto peptone, 10 mg L-1 FeSO4-7H2O, 3.2 mg L-1 MnSO4-H2O, 2.8 mg L-1 ZnSO4-7H2O, 4 mg L-1 CoCl2-6H2O, 200 mL L-1 Tween 80. The pH was adjusted to 5.8 and carbon source added before autoclaving. Pre-culture was cultivated for two days (in 1000 mL baffled Erlenmeyer flasks), after which mycelium was filtered and extensively washed with medium without any added carbon source. A total of 15 g of mycelium was divided equally between six 250 mL baffled Erlenmeyer flasks containing 50 mL basal liquid medium supplemented with either 10 g L-1 glucose, wheat bran or beechwood xylan as the sole carbon sources. Two independent duplicate experiments were performed for each substrate. After 4 h and 48 h of incubation at 50°C on a shaking incubator, RNA was extracted. RNA sequencing was performed with a 1:1 mixture of RNA samples isolated at 4 h and 48 h. The advantage of this strategy is that both early- and late-response genes are included and will be detected in the differential expression analysis. A potential drawback is, however, that the transcriptional response of genes induced only at the early or later time point will be diluted. Total RNA was extracted using TRIzol (Invitrogen) and chloroform, and further purified with the RNeasy Plant RNA Kit with on-column DNAse digestion. The quality of the purified RNA was verified by agarose gel electrophoresis, Nanodrop (Thermo Scientific) and Qubit (Life Technologies). The NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) was used to process the samples according to the manufacturer's instructions. Briefly, mRNA was isolated from total RNA using oligo-dT magnetic beads and used to synthesise cDNA. The cDNA was ligated with sequencing adapters and PCR amplified. The quality and yield after sample preparation were determined with the Fragment Analyzer (Advanced Analytical). The size of the resulting products was consistent with the expected size distribution (a broad peak between 300-500 bp).
attributes.tag: ENA first public
attributes.value: 2017-10-27
attributes.tag: ENA last update
attributes.value: 2017-10-24
attributes.tag: External Id
attributes.value: SAMEA104357873
attributes.tag: INSDC center alias
attributes.value: Chalmers University of Technology Department of Biology and Biological Engineering Division of Industrial Biotechnology
attributes.tag: INSDC center name
attributes.value: Chalmers University of Technology Department of Biology and Biological Engineering Division of Industrial Biotechnology
attributes.tag: INSDC first public
attributes.value: 2017-10-27T17:02:58Z
attributes.tag: INSDC last update
attributes.value: 2017-10-24T13:42:14Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6157:Wheat bran, replicate 1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: sample name
attributes.value: E-MTAB-6157:Wheat bran, replicate 1
attributes.tag: sampling time point
attributes.value: 1:1 mix of sample collected at 4 and 48 hours
attributes.tag: strain
attributes.value: FCH 10.5