GSM9352197: Porphyridium_residual_nitrate_0.5gL_rep2; Porphyridium purpureum; RNA-Seq
Biomass was flash-frozen and ground manually in liquid nitrogen. Total RNA was extracted using the TRIzol reagent protocol. Approximately 100 mg cells were homogenized in 1 mL TRIzol and incubated for 5 min at room temperature. Phase separation was performed by adding 0.2 mL chloroform per 1 mL TRIzol, followed by centrifugation (12,000 × g, 15 min, 4 °C). The aqueous phase was recovered and RNA precipitated with 0.5 mL isopropanol per 1 mL TRIzol. The pellet was washed with 75% ethanol, air-dried, and resuspended in RNase-free water. RNA integrity was verified on agarose gels. Stranded mRNA libraries were prepared using the Illumina TruSeq Stranded mRNA Library Preparation Kit following the manufacturer's instructions. Poly-A RNA was enriched, fragmented, reverse-transcribed, and converted to indexed cDNA libraries. Libraries were pooled and sequenced on an Illumina NovaSeq SP flow cell, generating paired-end 2 × 150 bp reads. Replicates correspond to independent technical sequencing libraries prepared from the same biological sample at each nitrate concentration.