title: polA enriched RNA-seq illumina sequencing data from axenic Epithemia clementina cultures, nitrogen depleted media, daytime
design: RNA isolation and sequencing  To capture a wide range of transcripts, axenic cultures of E. clementina were exposed to different nitrogen conditions and collected at different times in the day-night cycle. Axenic cultures of E. clementina were seeded in 175cm2 sterile vented flasks at a density of 1.2 million cells per flask. For conditions of nitrogen repletion, media in the flask contained 100M of ammonium. Cells were kept in -N or +NH4+ conditions for 72 hours and harvested two hours into the day period. Cells in nitrogen depleted conditions were additionally collected two hours into the night period. All cells were scraped from the flask, centrifuged to concentrate, resuspended in trizol, and flash frozen. Each condition was collected in triplicate for each experiment, and the whole experiment was performed twice. To lyse, the trizol suspended cells were held on ice and a sonicator probe was submerged at the center of the tube. Sonication was performed with 50/50 on/off pulses for one minute at an intensity setting of six. RNA was isolated using the QIAGEN RNeasy Plus Universal Mini Kit (74134) following the included protocol. 500ng of RNA per condition per replicate collected from the first experiment was used as input to the NEBNext poly(A) mRNA Magnetic Isolation module (E7490L) to enrich for mRNA and the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760L) and indexes from NEBNext Multiplex Oligos for Illumina Dual Index Primers Set 1 (E7600S) were used for library preparation. 350ng of RNA per condition per replicate collected from the second experiment was used as input to the Zymo-Seq RiboFree Universal cDNA Kit (R3001) and indexed with Zymo-Seq UDI Primer Set (Indexes 1-12) (D3008). For each experiment, libraries were pooled and sequenced on an Illumina NextSeq 2000 P3 for 2 x 150bp reads.