title: Ion Torrent Proton sequencing; Comparison of the gene expression profile of a wild type Pseudo-nitzschia multistriata strain and a strain displaying sex reversal
design: Comparison of the gene expression profile of a wild type Pseudo-nitzschia multistriata strain and a strain displaying sex reversal
library_construction_protocol: Wild type and transgenic cultures, grown at 18°C with 12:12 D/L, were collected during the late exponential phase of growth, in the light phase. The MRP3 CDS amplified from the cDNA of the MT+ strain LV149 was cloned downstream of the P. multistriata histone H4 promoter, producing the overexpression vector PmH4pMRP3At. This vector was transformed in the MT- strain LV136 using biolistic transformation, with the protocol described in Sabatino et al., 2015. The transformed strain which had incorporated the transgene was named LV136T3. 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. Transcriptome sequencing was conducted using the Ion P1 sequencing Kit v2 (Thermo Fisher Scientific) on an Ion Proton™ sequencer, following Manufacturer's instructions. Samples were run on two Ion P1 Chip Kit v3 chips (Thermo Fisher Scientific). All the libraries were sequenced in both chips to avoid the influence of batch effects.