The spheroids inoculated with mycelia of P. ostreatus were incubated at 25 °C for 3 months and were subsequently transferred into a cold room (16 °C) with high humidity (≥80%). The 3D clinostat used in this experiment has orthogonal X and Y-axes with two independent motors and is optimized for the 3D rotation of AZD2281 datasheet the substrate sphere used for mushroom cultivation. One such sphere was placed in the 3D clinostat, which was asymmetrically rotated (X-axis: 3.3 r.p.m., Y-axis: 3.9 r.p.m.) (Dedolfph & Dipert, 1971), and the other was firmly

fixed to the ground. After 2 weeks of cultivation in a cold room, the mature fruiting bodies of P. ostreatus were harvested from both spheroids. The total cellular RNA was extracted from the mature fruiting bodies using RNeasy® Midi/Maxi Handbook (Qiagen Inc.), and poly(A)+ RNA was prepared using an oligo(dT)-magnetic beads system (Takara Bio Inc.). We performed the cDNA synthesis according to the procedures reported in our

previous work (Miyazaki et al., 2005). cDNA-RDA was basically performed according to the procedures used in our previous work (Miyazaki et al., 2005). The synthesized double-stranded cDNAs derived from P. ostreatus fruiting bodies developed under simulated microgravity (clinostat-rotated) and static condition (fixed to the ground) were subjected to subsequent subtractive hybridization. For an isolation of Navitoclax datasheet upregulated genes under simulated microgravity, the cDNAs under clinostat-rotated and static conditions were used Carnitine dehydrogenase as a tester and a driver, respectively (Hubank & Schatz, 1994). To isolated downregulated genes under simulated microgravity, the cDNA under static and clinostat-rotated conditions were inversely used as a tester and a driver. After three repetitions of the subtractive steps with an alternation of the ligated oligonucleotides for PCR (Miyazaki et al., 2005), the finally subtracted cDNAs

were cloned and subjected to sequence analysis. Sequence analyses of the obtained genes were carried out on using the ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems). Preparations of sequencing samples were done according to the manufacturer’s protocol (Applied Biosystems). Computational homology searches were conducted using blastx and blastn (Altschul et al., 1997), utilities maintained by The National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi), The Broad Institute (http://www.broad.mit.edu/cgi-bin/annotation/fgi/blast_page.cgi), and DOE Joint Genome Institute (http://genome.jgi-psf.org/cgi-bin/runAlignment?db=Lacbi1&advanced=1). Semi-quantitative RT-PCR analyses were carried out according to protocols reported in our previous work (Miyazaki et al., 2007).