RNA-seq of Phaeodactylum tricornutum: HASP1 knockout day 17-3
For RNA extraction, 2e8 cells were harvested on the specified sampling day and pelleted at 6k RCF at 4C for 10 minutes, wherein the supernatant was gently poured off and the pellet was resuspended in 200 L TE buffer. This was then pipetted drop-wise into liquid nitrogen and ground into powder with a pre-chilled mortar and pestle. RNA extraction was performed with the Monarch Total RNA Miniprep Kit (New England Biolabs) according to manufacturers instructions. Purified RNA was quantified with a NanoDrop spectrophotometer and assessed for appropiate 260/230 and 260/280 ratios, and was run on a 1% agarose gel to confirm integrity. Libraries were prepared with the VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina according to manufacturer's instructions (Vazyme, NR603) and sequenced across five individual runs on a Illumina NextSeq on High Output for 75 cycles.