2.3. Library Preparation and Sequencing (Adherent Cultures)
Adherent CHO cell cultures in the log phase of growth were harvested for single-cell sequencing. A total of 2.5×106 cells were stained with 500 µL DMEM/F-12 containing L-glutamine (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) and 10 µg/mL Hoechst 33342 (Funakoshi, Tokyo, Japan) by stirring at 600 rpm for 40 min at 37°C. Cells were centrifuged for 5 min at 300×g and the supernatant was removed. Next, 500 µL of 1× PBS (no calcium, no magnesium, pH 7.2, Gibco; Thermo Fisher Scientific) was added to the cell pellet, which was pipetted up and down five times for thorough resuspension. Cells were centrifuged for 5 min at 300×g , the supernatant was removed, and the cells were thoroughly resuspended in 500 µL of 30% (w/v) Albumin D-PBS(−) Solution (fatty acid-free from bovine serum albumin; FujiFilm Wako Pure Chemical Corporation, Osaka, Japan) by pipetting the cell pellet up and down five times. Cells in the albumin-containing PBS were filtered with a 35-μm nylon mesh filter (Falcon, Corning, NY, USA). Cells were applied to the SH800S Cell Sorter and individual cells were sorted based on the Hoechst 33342-stained cell area of the FACS distribution. RNA isolation and complementary DNA (cDNA) synthesis were performed with the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech, Palo Alto, CA, USA) for sequencing according to the manufacturer’s protocol (Ziegenhain et al., 2017). Library preparation was performed with the Nextera XT DNA Library Prep Kit (Illumina) according to the manufacturer’s protocol. The finished cDNA libraries were quantified with the Agilent 2200 TapeStation system (Agilent Technologies, Santa Clara, CA, USA) and the QuantiFluor dsDNA System (Promega, Fitchburg, WI, USA) and then sequenced on an Illumina NextSeq 500 platform using 75-bp single-end reads. We performed single-cell RNA-seq analysis with 24 G1 cells and 8 G2 cells.