Illumina HiSeq 2000 sequencing; Gene expression changes at the onset of sexual reproduction in the marine planktonic diatom Pseudo-nitzschia multistriata
Gene expression changes at the onset of sexual reproduction in the marine planktonic diatom Pseudo-nitzschia multistriata
To induce the sexual phase, synchronized cultures of MT+ and MT- mating type were co-cultured in the bipartite glass apparatus where MT+ and MT- strains were physically separated by a 0.22μm pore size membrane filter that allowed chemical communication between them.Simultaneously, MT+ and MT- strains were grown in separate glass flasks as controls. Two and 6 hours after the co-culture started, samples (200 mL each) for RNA extraction were collected from the two strains in co-culture and from the parental strains cultivated in monoculture. Samples were collected onto 1.2μm pore-size membrane filters (RAWP04700 Millipore), placed in TrizolTM, flash frozen in liquid nitrogen immediately and stored at -80 0C until RNA extractions. RNA was extracted following the manufacturer’s instructions (Trizol reagent, Invitrogen) and the genomic DNA contamination was removed by DNase I treatment (RNase-Free DNase Set Qiagen) followed by RNA purification using RNeasy Plant Mini Kit (Qiagen). The quantity of RNA was determined by Qubit assay (Qubit® 2.0 Fluorometer, Life Technologies) and integrity was assessed by Bioanalyzer (2100 Bioanalyzer Instruments, Agilent Technologies) as well as by running samples on a 1.5% agarose gel. Libraries were prepared using a Beckman Biomek FX and the Illumina® TruSeq® Stranded Total RNA Sample Preparation kit, with poly-A selection and starting with 500 ng total RNA
Experimental Factor: MT+
mating type
Experimental Factor: membrane separated co-culture with MT- strain (B857)
growth condition
Experimental Factor: 6
sampling time point