Vitronectin xf coated

Human-induced pluripotent stem cells (hiPSCs) (https://www.hipsci.org/lines/#/lines/HPSI0714i-nufh_3, https://www.hipsci.org/lines/#/lines/HPSI0914i-euts_1)^{59 (link)} were thawed and cultured under feeder-free conditions on Vitronectin XF™-coated (Stem Cell, #07180) 10 cm tissue culture treated plates (Corning) in complete Essential 8 medium (Life Technologies #A1517001) supplemented with 1% Penicillin/Streptomycin (Invitrogen, #15140122). Cells were incubated at 37°C, 5% CO2 and media changed every day except for the day of passaging. Cells were routinely passaged using 0.5 mM EDTA (Life Technologies #AM9260G) at least 3 times post-thawing before collection. HiPSCs were harvested using Accutase (Millipore, #SCR005) to generate a single cell suspension. Single cells were resuspended in 1X PBS (Thermo Fisher Scientific, #10010023) and passed through a 40uM filter (Corning #CLS431750-50EA) before FAC sorting (BD Influx™ Cell Sorter) into 96-well plates (Framestar, #4TI-0960) containing 2 μl lysis buffer with 1 U/μl Rnase Inhibitor (RRI) and ERCC spike in mix at a 1:20 dilution (Ambion Cat #4456740). Plates were promptly sealed (Thermo Fisher Scientific #AB0626), spun down and frozen at -80 °C until further processing.

Human H9 embryonic stem cells (WiCell WA09) were cultured at 37°C, 5% CO₂ in Essential 8 Flex medium on Vitronectin-XF–coated (STEMCELL Technologies) plates. Cells were passaged every 3 to 4 days based on cell density. Cells tested negative for mycoplasma contamination throughout the study. All DNA libraries were quantified by quantitative PCR using the KAPA Library Quantification Kit for Illumina Platforms (Roche) and were tested for quality and primer and adapter contamination using Agilent 2100 Bioanalyzer High Sensitivity DNA chip before proceeding to high-throughput sequencing.