title: WT1
organism: Aspergillus fumigatus
description: Protocols: Total RNA was isolated from wild-type and mpkA strains cultured in Aspergillus minimal medium (AMM) for 16 h at 37 C (Jain et al., 2011), using the Qiagen RNeasy Plant Mini kit (Qiagen, Germany), according to the manufacturers instruction. Transformation of A. fumigatus KU80D with plasmid pCR2.1DmpkA/Ura to generate the mpkA deletion mutant was carried out using protoplasts as previously described (Weidner et al., 1998). Transformation of A. fumigatus strain DmpkA for complementation of the deletion was per- formed as follows: conidia of the DmpkA strain were plated on AMM agar plates supplemented with 1 g/L 5-fluoroo- rotic acid (5-FOA, Fermentas, Germany) and 10 mM uracil (Fluka, Germany), in order to obtain DmpkA pyrGA strains. DmpkA pyrGA colonies were screened by Southern blot analysis for loss of the pyrG gene. Plasmid delta mpkA/Ura was used to transform DmpkA pyrGprotoplasts. Transformants were analyzed by PCR and Southern blot analysis for mpkA complementation. A posi- tive transformant strain was designated mpkAc. Plasmid pUCPyrG2mpkAp-lacZ, containing the mpkA promoter fused in frame with the E. coli lacZ gene, was used to transform A. fumigatus strain KHDpyrG (Sugareva et al., 2006). Southern blot analysis of the resulting trans- formant strain MpkAp-lacZ indicated the presence of the gene fusion in single copy at the pyrG gene locus. (Valiante V, Heinekamp T, Jain R, Haertl A, Brakhage AA: The mitogen-activated protein kinase MpkA of Aspergillus fumigatus regulates cell wall signaling and oxidative stress response. Fungal Genet Biol 2008, 45: 618-627.) Total RNA was isolated from wild-type and mpkA strains. Total RNA was used for Illumina next-generation sequencing (Bentley et al., 2008). For library preparation 5ug of total RNA per sample were processed using Illumina mRNA-Seq sample prep kit (RS-100-0801) following the manufacturers instruction
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2012-09-30T17:02:28Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2012-08-02T12:55:16Z
attributes.tag: External Id
attributes.value: SAMEA1523776
attributes.tag: INSDC center name
attributes.value: Leibniz-Institute for Natural Product Research and Infection Biology Hans Knoll Institute
attributes.tag: INSDC first public
attributes.value: 2012-09-30T17:02:28Z
attributes.tag: INSDC last update
attributes.value: 2012-08-02T12:55:16Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-1236:WT1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: geneticmodification
attributes.value: transformation
attributes.tag: genotype
attributes.value: wild_type
attributes.tag: sample name
attributes.value: E-MTAB-1236:WT1
attributes.tag: scientific_name
attributes.value: Aspergillus fumigatus