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.