E-MTAB-7463:AGHEt3_s
Illumina HiSeq 4000 sequencing; RNAseq of Ustilago maydis with inducible clp1 and Δcib1
RNAseq of Ustilago maydis with inducible clp1 and Δcib1
AGHEt3_s
For RNAseq, strains were inoculated in YNB (pH 5.8) supplemented with 1% glucose and 0.2% ammonium sulfate (YNBG) overnight to an OD600 of 0.25 and shifted to YNB (pH 5.8) supplemented with 1% arabinose and 0.2% ammonium sulfate to induce Clp1 expression (Pcrg1:clp1). To induced the UPR, TM was added to a final concentration of 5 µg/ml and cells were further incubated for 4 hours, at 28°C. Cells were harvested and quick frozen in liquid nitrogen.  RNA was prepared from cells during exponential growth using Trizol reagent (Invitrogen) according to the manufacturer's instructions (Scherer et al., 2006), followed by removal of residual DNA with TurboDNase (Ambion/Lifetechnologies). If RNA was used for RNAseq anaylsis samples were further column-purified with the RNeasy Kit (Qiagen). Integratity ofRNA was checked by ethidium bromide staining and by Bioanalyzer equipped with an RNA 6000 Nano LabChip kit (Agilent). NEB Next Poly(A) mRNA Magnetic Isolation Module (New England BioLabs, Frankfurt am Main, Germany) was used to reduce the amount of rRNA-derived sequences. For sequencing, the strand-specific cDNA libraries were constructed with a NEBNext Ultra directional RNA library preparation kit for Illumina (New England BioLabs, Frankfurt am Main, Germany). To assess quality and size of the libraries samples were run on an Agilent Bioanalyzer 2100 using an Agilent High Sensitivity DNA Kit as recommended by the manufacturer (Agilent Technologies, Waldbronn, Germany). Concentration of the libraries were determined using the Qubit® dsDNA HS Assay Kit as recommended by the manufacturer (Life Technologies GmbH, Darmstadt, Germany)
Philipps University   Institute for Molecular Biology and Tumor Research
E-MTAB-7463:AGHEt3_s
Experimental Factor: compound
none
Experimental Factor: genotype
wild type genotype