Illumina HiSeq 2000 paired end sequencing; Gene expression analysis of monokaryotic and dikaryotic mycelia from Coprinopsis cinerea
Gene expression analysis of monokaryotic and dikaryotic mycelia from Coprinopsis cinerea
To study the impact of the mating type locus on the transcriptional profile of C. cinerea, A43mutB43mut and Okayama7 mycelia (ABVM24 and O7VM24) were grown in triplicate on YMG plates covered with cellophane discs at 37C in the dark for 96 h and 120 h, respectively, and transferred to 24C for 72 h in the dark. Mycelia were harvested, flash frozen and stored at -80C. From each ABVM24C replicate or O7VM24C replicate , 20 mg lyophilized material were lysed in three FastPrep FP120 homogenization steps of 45 s at 4.5, 5.5 and 6.5 m/s in the presence of 250 mg 0.5 mm glass beads, cooling the samples for 5 min on ice between steps. RNA was extracted using 1 mL Qiazol (Qiagen) and 0.2 mL chloroform ReagentPlus (Sigma-Aldrich). The solution was centrifuged at 12000 xg for 15 min at 4C; thereafter, RNA from the resultant aqueous phase was washed on-column using the RNeasy Lipid Tissue Mini Kit (Qiagen) and eluted in 60 uL RNase-free water. Concentration and integrity of the purified RNA were determined with a Qubit (1.0) fluorometer (Life Technologies) and a Bioanalyzer 2100 (Agilent), respectively. Samples with a 260/280 nm ratio of 1.8-2.1 and a 28S/18S ratio of 1.5-2 were later used in library construction. TruSeq Stranded mRNA Sample Prep Kit (Illumina) was used to construct ABVM24 and O7VM24 expression libraries (three biological replicates each). Briefly, total RNA (1 ug) was poly(A) enriched and then reverse-transcribed into double-stranded cDNA in the presence of Actinomycin during first-strand synthesis. Double-stranded cDNA was fragmented, end-repaired and polyadenylated before being ligated to TruSeq adapters and selectively enriched by PCR. Concentration and quality of the enriched libraries were assessed using Qubit (1.0) fluorometer and LabChip GX (PerkinElmer), showing an average fragment size of 260 bp. Libraries were normalized to 10 nM with 10 mM Tris-HCl pH 8.5 supplemented with 0.1% Tween 20. In addition, TruSeq PE Cluster Kit v3-cBot-HS (Illumina) was used to generate clusters from 10 pM pooled normalized libraries.
INSTITUTE OF MICROBIOLOGY, ETH ZURICH
E-MTAB-1993:O7VM24C_replicate_2
Institute of Microbiology, ETH Zurich
E-MTAB-1993:O7VM24C_replicate_2
Experimental Factor: strain
Okayama7