scRNA-seq of cells: Mono_iPSC1_D8
The DYR0100 cell line (“iPSC1” hereafter) was kindly provided by the Stem Cell Bank of the Chinese Academy of Sciences (Cat# SCSP-1301, CSTR: 19375.09.3101HUMSCSP1301). The monoclonal iPSC1 line was generated by single-cell dissociation and limiting dilution of iPSC1, followed by manual selection and expansion of individual colonies. Monoclonal iPSC1 cells were passaged with Accutase and replated in a Matrigel-coated 6-well plate with Essential 8 medium containing 10 μM ROCK inhibitor. Lentiviruses overexpressing rtTA and Nfib/Sox9 were added to each well at a multiplicity of infection of 10 on the same day. The medium was daily replaced by Essential 8 medium. When the iPSCs reached 80% confluence (designated as Day 0), the medium was substituted with 2 mL of fresh Essential 8 medium containing 1 μg/mL doxycycline (Sigma-Aldrich, D9891).On Day 1 and 2, the medium was exchanged with 2 mL expansion medium. On Day 3, the medium was replaced by 2 mL expansion medium and FGF medium (3:1 v/v). On Day 4, the medium was substituted with 2 mL expansion medium and FGF medium (1:1 v/v). On Day 5, the cells were washed by DPBS (without Ca2+/Mg2+) (Thermo Fisher Scientific, 14190250) and dissociated using 500 μL Accutase for 30 min at 37°C. The progression of cell detachment was continuously monitored under a microscope. Cell suspensions were collected and centrifuged at 300 × g for 5 min to remove as much supernatant with Accutase as possible without disturbing the cell pellet. Then, 3–4 × 105 cells per well were evenly replated in a 6-well plate pre-coated by Matrigel with 2 mL expansion medium and FGF medium (1:3 v/v). On Day 6 and 7, the medium was exchanged daily with 2 mL FGF medium. Doxycycline (1 μg/mL) and puromycin (5 μg/mL, Thermo Fisher Scientific, A1113803) were kept in the medium throughout the experiments from Day 1–7. Eight days after doxycycline induction, cells on Day 8 were collected for scRNA-seq. The cultured cells were pre-washed with DPBS and dissociated with 200 µL Accutase in a 6-well plate for 10 min at 37°C. The progression of cell detachment was constantly monitored under a microscope. Cell suspensions were collected and centrifuged for 5 min at 300 × g to remove as much Accutase as possible without disturbing the cell pellet. The cells were resuspended in DPBS with 0.04% filtered bovine serum albumin and 60 U/mL RNasin Plus Ribonuclease Inhibitor (Promega, N2615). After quantification by a Countess Automated Cell Counter (Invitrogen, C10281), cell suspensions were diluted to 700–1,200 cells/µL on ice for subsequent scRNA-seq library preparation. A Chromium Next GEM Single Cell 3′ Reagent Kit v3.1 and Gel Beads Kit (10x Genomics) were used following the manufacturer's guidelines. Briefly, single-cell suspensions, gel beads, as well as partitioning oil were added to the 10x Genomic Chromium Chip (Next GEM chip G) and subjected to the 10x Chromium Controller device, which encapsulated single cells within individual Gel Beads-in-emulsion (GEMs). The targeted number of cells was 10,000 in each sample. Captured cells were lysed, and the transcripts inside the individual GEMs were barcoded through reverse transcription. Constructed 10x libraries were quantified by a Qubit 4 Fluorometer (Invitrogen) with a Qubit 1x dsDNA HS Assay Kit (Invitrogen). Quality control for the 10x libraries was performed using a Fragment Analyzer 5200 (Agilent) with a DNF-474 HS NGS Fragment Kit (Agilent). Sequencing of libraries was performed on an Illumina NovaSeq 6000 Sequencing platform (Novogene), with a paired-end read length of 150 bp and 100 GB raw data per sample.