NextSeq 500 sequencing; scRNA-seq of mouse EpiSCs (epiblast stem cells) differentiated to extraembryonic mesoderm in vitro, in a monolayer
scRNA-seq of mouse EpiSCs (epiblast stem cells) differentiated to extraembryonic mesoderm in vitro, in a monolayer
EpiSCs were seeded at ~6 × 103 cells/cm2 in six-well cell-culture plates pre-coated with 15 µg/ml human Fibronectin, and differentiated to PS as described in the article.  At desired time-points, cells were dissociated using Accutase (Thermo Fisher Scientific, 00-4555-56) for 3 min at 37 °C and counted. 500,000 cells per condition were washed with ice-cold FACS buffer (10% (v/v) FBS in PBS) and resuspended into 1 ml ice-cold FACS buffer containing 1 µg/ml DAPI.Cells were washed with 500 μl ice-cold FACS buffer, centrifuged again at 350 × g for 3 min and resuspended into 250 μl ice-cold FACS buffer containing 1 µg/ml DAPI. Singlecells from in vitro-differentiated cells were sorted into Eppendorf Polypropylene U-shaped 384-well Twin Tec PCR Microplates (Thermo Fisher Scientific, 10573035), containing 2 μl of lysis solution (0.2% (v/v) Triton X-100) supplemented with 0.4 U/μl RNasin Ribonuclease Inhibitor (Promega, N2515) and 400 nM indexed RT primer from group 1 (1–96 barcodes) or group 2 (97-192 barcodes) Cells were washed with 500 μl ice-cold FACS buffer, centrifuged again at 350 × g for 3 min and resuspended into 250 μl ice-cold FACS buffer containing 1 µg/ml DAPI. Singlecells from in vitro-differentiated cells were sorted into Eppendorf Polypropylene U-shaped 384-well Twin Tec PCR Microplates (Thermo Fisher Scientific, 10573035), containing 2 μl of lysis solution (0.2% (v/v) Triton X-100) supplemented with 0.4 U/μl RNasin Ribonuclease Inhibitor (Promega, N2515) and 400 nM indexed RT primer from group 1 (1–96 barcodes) or group 2 (97-192 barcodes). Additionally, 71 WT EpiSCs were sorted into each plate, as spike-in control for batch-effect correction. Capture plates were prepared on the Bravo automated liquid handling robot station (Agilent) using 384-filtered tips (Agilent, 19133-142). Index sorting was performed using a FACS Aria III cell sorter (BD Biosciences) at the DanStem Flow Cytometry Platform (University of Copenhagen, Copenhagen, Denmark), sorting live cells as singlets. Immediately after sorting, plates were spun down, snap-frozen on dry ice and stored at −80 °C until further processing. Semi-automated library preparation was performed, using 10–12 total cycles of PCR amplification and AMPure XP beads (Beckman Coulter Life Sciences, A63881) for purification63. DNA concentration was measured with a Qubit Fluorometer (Thermo Fisher Scientific, Q32854) and fragment size was determined with a Fragment analyzer (Agilent).