Non-cell-autonomous control of gastruloid development by the lncRNA T-UCstem1 2 through DKK1-dependent modulation of WNT signalling
Non-cell-autonomous control of gastruloid development by the lncRNA T-UCstem1 2 through DKK1-dependent modulation of WNT signalling
Gastruloid formation assay was performed as described (Baillie-Johnson et al., 2015; Cermola et al., 2021). Naïve ESCs were cultured at low density (250 cells/cm2) and the resulting colonies were dissociated with accutase (Sigma-Aldrich; cat: A6964; 3 min at 37°C). Cells were seeded in N2B27 at 1.0-2.5 x 102 cells/well (40 μL) in ultra-low attachment 96 multi-well plates (Corning Costar; cat: 7007) and allowed to aggregate. At 48 h AA, CHIR was added (3 μM) to the culture medium and maintained for 24 h. From 72 h onward, the medium (150 μL) was refreshed daily up to 120 h. DKK-1 recombinant protein (200 ng/mL) and WAY262611 (0.1, 0.20, 0.25, 0.50, 0.75 and 1μM) were added during throughout the assay. Gastruloids from NT and T-UCstem1 KD-1 were collected after 120 h. gastruloids were collected in 15 mL tubes, pelleted by gravity and dissociated by trypsin incubation at 37 ̊ for 7 min, washed with PBS and resuspended in cold PBS supplemented with 0.1% BSA. The single-cell suspensions were then visually inspected for determining cell number and viability as well as the absence of residual cellular aggregates and finally loaded into the Chromium Controller (10X Genomics) to generate single cell GEMs. After GEM generation, all the steps of cDNA synthesis, clean-up and amplification, DNA fragmentation, end-repair, adapter ligation and indexing PCR-amplification were done with the Chromium Single Cell 3' reagents following the manufacturer's protocol while the DNA purification as well as the size selection steps were done with SPRIselect magnetic beads (Beckman Coulter, Brea, CA) as indicated by the 10X protocol. Finally, the concentration of each indexed library was determined by using Qubit dsDNA BR Assay (Thermo Fisher Scientific, USA) and their quality were assessed with the D1000 screentape on the TapeStation 4150 (Agilent Technologies, Santa Clara, CA).