title: Comparative single cell RNA seq of gastruloids from mESCs grown in 3 distinct media conditions facilitating distinct pluripotent states at 3 different developmental time points
design: Comparative single cell RNA seq of gastruloids from mESCs grown in 3 distinct media conditions facilitating distinct pluripotent states at 3 different developmental time points
library_construction_protocol: Mouse ESCs (mESCs) and gastruloids were maintained and generated as previously described (see Anlas, Baillie-Benson et al. 2021, Programmed Morphogenesis: Methods and Protocols, p131-147). In brief: mESCs were cultured in either DMEM-based Serum-LIF (SL), SL + 0.3uM PD03 (SL + 1i), or SL + 1.5uM CGP77 + 3uM CHIR99 (SL + a2i) . Culture entailed passaging every 2-3 days using TrypLE. For Gastruloid generation, mESCs were detached using TrypLE, cell counting was performed and the desired number of cells (usually ~300/well) were plated in 40ul N2B27 into all wells of a round bottom, low adhesion 96-well plate. After incubating for 48h in case of mESCs from SL and SL + 1i, 150ul N2B27+3uM CHIR99 (Wnt agonist) were added per well, in case of SL + a2i, at 48h 150ul only N2B27 were added and the CHIR99 pulse was performed at 72h (via exchanging 150ul of the media per well). 150ul N2B27 were exchanged each developmental day until gastruloids were used for further experimental procedures. Samples were extracted from wells of low-adhesion, round-bottom 96-well plates into a 15ml centrifuge tube via a P1000 pipette with wide-bore RNAse-free filter tips or via organoid collection plates. Once all samples were collected, thus yielding one 15ml centrifuge tube per condition, aggregates were centrifuged at 180g for 2min. Hereafter, the supernatant was removed and 1ml TrypLE (ThermoFisher Scientific, 12604) was added per tube for dissociation and samples were incubated for 5min at (37C, 5% CO2). Then, TrypLE was deactivated using 4ml ESL and gastruloids were centrifuged for 2min at 180g. Supernatant was removed, samples were washed with 5ml cold (4C) 0.1% RNase-free BSA (ThermoFisher Scientific, AM2616) in DPBS−/− (BPBS) and centrifuged (2min, 180g). From this step onwards, samples were maintained at 4C. Following another washing step and removal of the supernatant, the pellet was resuspended in 50-100ul cold BPBS (depending on pellet size) by gently pipetting up and down 3-5x with a P200 tip. The cell suspension was then filtered through a 35um filtered cap tube (Corning, 352235). Cells were counted as previously described,aiming for a concentration of around 1,000,000 cells/ml. In case of high concentrations (above 1,500,000cells/ml) , the cell suspension was diluting with cold BPBS and re-counted. For the 0h timepoint, mESCs were trypsinized using 1ml Trypsin-EDTA 0.05%. Following neutralization (4ml ESL) and centrifugation (180g, 3min), the supernatant was discarded and cells were washed with 5ml cold BPBS. Cells were then kept on ice, until samples from other experimental conditions reached this washing step. At this point, all samples were joined together for centrifugation and further procedures illustrated above. cDNA libraries were constructed with Chromium Next GEM Single Cell 3' Library & Gel Bead Kit v3.1 (10x Genomics) according to manufacturer's protocol.