RNA-seq of clonal human pluripotent stem cell lines and control human pluripotent stem cell lines
RNA-seq of clonal human pluripotent stem cell lines and control human pluripotent stem cell lines
To prepare RNA for sequencing, cells were dissociated using accutase, diluted in DMEM/F12 and centrifuged to pellet the cells. Supernatant was aspirated and the resulting pellet was flash frozen on dry ice and stored at -70°C until all samples were collected. Cells were cultured throughout in a humidified incubator with 5% O2 and 7% CO2 at 37°C. Established nPSCs were maintained on inactivated mouse embryonic fibroblasts in t2iLGöYXaa (0.5 μM CHIR99021, 1 μM PD0325901, 2.5 μM Gö6976, 10 μM Y-2763210 ng/ml LIF, 125 μM ascorbic acid, 2μM XAV939) or PXGL (1 µM PD0325901, 2 µM Gö69832,  2 µM XAV939  10 ng/ml human LIF in N2B27). Inhibition of ROCK (10 μM Y-27632) was performed during plating of cells in PXGL. Both t2iLGöYXaa and PXGL conditions are accompanied by a Geltrex spike-in (20 μL of 1:4  in DMEM/F12). Cells were passaged using Accutase. Conventional primed PSCs were cultured in N2B27 supplemented with ACTIVIN-A (20 ng/mL), FGF2 (12.5 ng/mL), and XAV939 (2 µM) (AFX) on Geltrex pre-coated plates (1:100 in DMEM/F12, 1 h at 37°C) and passaged using 0.5 mM EDTA in PBS. RNA was collection using TRIzol (ThermoFisher 15596026) followed by column purification with the PureLink RNA mini kit (ThermoFisher 12183020) In brief, pellets 1mL Trizol added and pipetted up and down and transferred to a 1.5ml Eppendorf tube. Chloroform (20μl) was added and the tubes incubated at RT for 2-3min. The samples were centrifuged for 15min at 12000g at 4°C. We kept both the upper aqueous phase containing RNA, and the lower phenol phase, containing the protein. To the aqueous phase, equal volumes of 70% ethanol and mixed by vortexing. This was then purified using the PureLink RNA mini kit, following the kit instructions. The sample was bound to the column, and oncolumn DNase treatment was carried out PureLink DNase set (12185010) to remove any contaminating genomic DNA. After washing and drying the column, the RNA was eluted with 2 x 50μl RNase-free water, and the samples were stored at -70°C.  Initial quality control of the RNA was performed with Agilent RNA tapestation reagents (5067-5576; 5067-5577; 5067-5578) and the Qubit RNA HS Assay Kit (Q32855). Sample library production was performed using 500ng of total RNA with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760) and the NEBNext Multiplex Oligos for Illumina (E6440), with 96 unique dual index primer pairs, in conjunction with NEBNext Poly(A) mRNA 15 Magnetic Isolation Module (E7490). This was performed according to manufacturer's instructions. Quality control of the resulting libraries was performed with Qubit dsDNA HS Assay Kit (Q32854) and Agilent DNA 5000 tapestation reagents (5067-5588; 5067-5589).