2.2. Library Preparation and Sequencing (Serum-free Suspension Cultures)
After days 4 (D4), 8 (D8), and 11 (D11), the CHO cells in three replicate serum-free suspension culture flasks were harvested for single-cell sequencing, and the remainder was discarded. Samples of the CHO cells in suspension culture were directly applied to the SH800 Cell Sorter (Sony, Tokyo, Japan), and individual cells were collected directly into PCR tubes as previously reported (Hayashi et al., 2010). A total of 0.5×106 cells were stained with 1 mL phosphate-buffered saline (PBS) containing 10 µg/mL Hoechst 33342 at 37°C for 15 min. The stained cells were sorted based on the Hoechst 33342-stained cell area of the fluorescence-activated cell sorting (FACS) distribution (Hayashi et al., 2010). Library preparation for single-cell Quartz-Seq was performed as previously reported (Sasagawa et al., 2013). We prepared a library for conventional RNA-seq using a commercial kit (TruSeq RNA Library Prep Kit; Illumina, San Diego, CA, USA) in accordance with a previous study (Yamano-Adachi et al., 2019). Conventional RNA-seq and single-cell Quartz-Seq were performed with a commercial sequencer (NextSeq 500; Illumina) in accordance with the manufacturer’s protocols. We performed single-cell Quartz-Seq as previously reported (Sasagawa et al., 2013) on 85 D4, 70 D8, and 78 D11 CHO cells. The resulting short-read data have been deposited in the Short Read Archive of the DNA Data Bank of Japan (DDBJ) under project ID DRA004159.