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.