Rnase inhibitor 2313a

To evaluate the effects of medium components on cells, we analysed changes in cell morphology and gene expression by adding or removing a component based on Chem. We added CHIR99021 (S2924, Selleckchem), valproic acid (VPA, S3944, Selleckchem), or both CHIR99021 and VPA to the culture cells, respectively. The cells were dissociated into single‐cell suspensions and stained with 7‐AAD viability staining solution (420403, BioLegend) for fluorescence‐activated cell sorting (FACS; BD FACS ARIA SORP, BD Biosciences) on Days 6 and 14. Wnt3A, R‐spondin1, Noggin, A83‐01 or EGF were removed from Chem. to form minus culture medium. As above mentioned, the cells were digested for FACS on Days 3, 6 and 10. 7‐AAD⁻ cells were sorted into strip tubes with 8 μl lysis buffer by FACS. The lysis buffer contained 1 U/μl RNase Inhibitor (2313A, Takara), 0.475% Triton X‐100, 2.5 μM oligo dT primer and 2.5 mM dNTP (4019, Takara). There were three replicates per sample for subsequent bulk RNA sequencing library construction.

The cells at T0 were digested with 0.05% Trypsin-EDTA at 37°C for 5 min. For T3, T6, or T9, we digested the cells with 0.05% Trypsin-EDTA at 37°C for 5–10 min after the digestion of the cells with 0.02% collagenase at 37°C for 3–5 min. We obtained single cells by pipetting the digested cell sheets. Dissociated cells were resuspended in washing buffer consisting of 2.5% FBS in PBS, counted, and stained with Hoechst 33342 (H-1399; Invitrogen) and SytoxRed dead-cell stain (S34859; Thermo Fisher Scientific). Hoechst positive/Sytox negative single cells were sorted into 1 µl of cell lysis buffer consisting of 0.3% NP40 (28324; Thermo Fisher Scientific), 0.12 dNTPs (N0477; New England BioLab, Ipswich, MA, United States), 1U RNase Inhibitor (2313A; TaKaRa Bio), and 0.11 µM 384 well-unique reverse transcription primer in a 348-well PCR plate (0030129547; Eppendorf, Hamburg, Germany).