title: RNA-seq of cricetulus griseus (CHO-S)
design: After a four-day growth period in the 250 mL shake flasks 5 million CHO cells were centrifuged at 500 rcf for 5 minutes and stored in RNA-later at -80 degrees celcius. RNA was extracted from cell lines stored in RNA-later using the RNeasy Mini Kit (Qiagen). The complete protocol is available online at (https://www.qiagen.com/us/resources/resourcedetail?id=14e7cf6e-521a-4cf7-8cbc-bf9f6fa33e24&lang=en). The essential steps are summarised below: Cells were thawed to room temperature and resuspended in 600 l of Buffer RLT. Roughly 150 l of 0.1 mm zirconia beads (Thermo Fisher Scientific) were added to the cell suspension for homogenisation, followed by beating using the Bead Ruptor Elite (Omni. Inc) for 10 minutes. The beads were pelleted through centrifugation at 1,000 g for 5 minutes at 4C. Supernatant was transferred to a clean 1.5 ml Eppendorf tube, mixed with an equal volume of 70% ethanol, and then loaded into the RNeasy spin column. The column was centrifuged at >8,000 g for 15 seconds, a step that was repeated until the entire sample had been processed. The column was then washed with 700 l of buffer RWI followed by 500 l of buffer RPE, each step followed by centrifugation. Lastly, RNA was eluted with 50 l of RNase-free water (Qiagen). RNA quality and concentration were assessed using an Agilent 2100 bioanalyzer and an RNA Nano Chip with RNA 6000 NanoLadder (Agilent Technologies). Samples from all cell lines were sent to the Ramaciotti Centre for Genomics (UNSW) for further processing and sequencing. Libraries were prepared from total RNA using the Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus kit (Illumina). These libraries were PCR amplified and loaded onto a single NovaSeq 6000 S4 lane, multiplexing all 16 samples, for 150 base pair paired-end sequencing. This produced approximately 120M read pairs per sample.