MARS-seq Version 2
MARS-seq Version 2
Cells were collected in TC lab, and Eomes-GFP were sorted by FACS Single cells from either in vitro-differentiated cells or transgenic embryos 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) as described in Mariani et al., 2021. Capture plates were prepared on the Bravo automated liquid handling robot station (Agilent) using 384-filtered tips (Axygen, 302-82-101). Index sorting was performed using either a FACS Aria III cell sorter (BD Biosciences) or a SONY SH800S cell sorter (Sony Biotechnology) at the DanStem/reNEW Flow Cytometry Platform (University of Copenhagen, Denmark), gating in SSC-A versus FSC-A to collect live cells, and then in FSC-W versus FSC-A to sort only singlets. For in vivo experiments, only GFPpos cells were sorted to capture Eomes-expressing cell types. 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 as described in Jaitin et al., 2014, using 10-12 cycles of PCR amplification and AMPure XP beads (Beckman Coulter™Agencourt: A63881) for purification. DNA concentration was measured with a Qubit Fluorometer (Thermo Fisher Scientific, Q32854), and fragment size was determined with a Fragment analyzer (Advanced Analytical).