title: GSM9269838: SU016WT-24h-S2; Chromochloris zofingiensis; RNA-Seq
library_construction_protocol: For RNA extraction, cells were collected at different time points by centrifugation (3200 g, 5 min at 4oC) and frozen in liquid nitrogen. RNA extraction and purification was performed as previously described with certain modifications (6). Briefly, 50 mL culture was pelleted via centrifugation (3,200 g, 5 min, 4°C) and the pellet was flash frozen in liquid nitrogen followed by fragmentation with a spatula on dry ice. Cells are then suspended in cold ethanol, transferred to Lysing Matrix D tubes, homogenized using the MP Biomedicals FastPrep-24TM 5G homogenizer (60 s, 6.5 m/s, 3 cycles), and lysed in fresh buffer containing SDS/proteinase K. The lysate is clarified by centrifugation, mixed with TRIzol (Thermo Scientific) and centrifuged to separate phases; the aqueous phase is combined with ethanol and loaded onto miRNeasy columns (Qiagen) under vacuum. After on-column DNase digestion (15 min, 30°C) RNA was eluted in water, precipitated with sodium acetate and ice-cold ethanol, incubated (−20°C, 30 min), and pelleted by centrifugation. The RNA pellet is washed with 70% ethanol, air-dried, and resuspended in RNase-free water. Finally, RNA quality is assessed via NanoDropTM (Thermo Scientific) spectrophotometry and a 2100 Bioanalyzer (Agilent) (RIN > 8, distinct 18S/28S peaks). Around 3 µg RNA was submitted to Joint Genome Institute in NovoSeq S4 plates. Further steps involved creation of polyA selection-based cDNA library for 2X151 paired end read sequencing using Illumina NovoSeq. Following quality trimming of reads using the in-house JGI program BBDUK, samples had an average rRNA contamination of 0.3% and a genome mapping rate of 99.57%.