title: NextSeq 500 paired end sequencing; Single-cell RNA-seq of tailbuds from control, Pbx1/Pbx2 compound and Pbx1/Pbx2 double-knockout E8.5/E9.0 embryos.
design: Single-cell RNA-seq of tailbuds from control, Pbx1/Pbx2 compound and Pbx1/Pbx2 double-knockout E8.5/E9.0 embryos.
library_construction_protocol: Tailbuds were dissected from E8.5/E9.0 embryos and dissociated into 300 μl warmed Trypsin 0.05% (w/v) EDTA for 10 min at 37°C. Digestion was stopped by addition of 600 μl ice-cold FACS buffer followed by centrifugation at 350 x g for 3 min. Cells were washed with 500 μl ice-cold FACS buffer, centrifuged again at 350 x g for 3 min and resuspended into 250 μl ice-cold FACS buffer containing 1 µg/ml DAPI.  Single-cells from embryonic tailbuds 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) (Jaitin D.A. et al., Science, 343, 776-779 (2014)). Additionally, 71 wild-type EpiSCs were sorted into each plate, as spike-in control for batch-effect correction. Two wells were left empty in each plate, as a no-cell control during data analysis. 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 as described in Jaitin D.A. et al., Science, 343, 776-779 (2014), using 10-12 total cycles of PCR amplification and AMPure XP beads (Beckman Coulter Life Sciences, A63881) for purification. DNA concentration was measured with a Qubit Fluorometer (Thermo Fisher Scientific, Q32854) and fragment size was determined with a Fragment analyzer (Agilent).