GSM8722492: Multiplexed scRNA-seq from Gastruloids of various size at 120h rep2; Mus musculus; RNA-Seq
Gastruloids seeded from different cell numbers (100, 300, 600, 1800, 5400 cells) were grown until 120 h and 144 h across two independent replicates. For each condition the number of gastruloids used was chosen to unsure that a minimum of 200 000 cells were obtained for each sample and a minimum of 24 gastruloids were collected for each sample to limit the impact of gastruloids-to-gastruloids variation. Gastruloids were collected and washed in 1ml of PBS in a 1.7 ml Eppendorf tube and dissociated using 100μl of Accutase (Stempro) for 5 minutes at 37°C. Full dissociation was verified to ensure absence of doublets and if necessary, it was completed using mechanical dissociation by pipetting. All centrifugation were done at 400 g for 5 minutes. Conditions were multiplex using the CellPlex procedure according to manufacturer's recommendations. Cells were incubated in 50μl of cell multiplexing oligos (3' CellPlex Kit Set A, PN-1000261) for 5 minutes at room temperature. They were then thoroughly washed three times with 1 ml PBS 1% BSA ensuring to remove as much as possible of the supernatant each time to prevent sample-to-sample contamination. Each sample was then counted and viability was assessed using a Countess 3 automated cell counter (Invitrogen) and viability was above 90% in all cases. Samples were then pooled in desired proportion to ensure proper representation of each experimental condition and the pooled cell suspension was filtered using a 40 μm cell strainer (Flowmi, BAH136800040). The final count was performed and 24 000 cells were targeted for recovery using the 10x Genomics approach following their recommendations since multiplexing allows for the resolution of more doublet cells, yielding on average 15000 singlet cells that can be used for analysis. The pool of cells was counted and subjected to single cell RNA-seq following manufacturer's recommendation (protocol CG000388 Rev A). We aimed for a recovery of c.a. 24000 cells per sample as a large number of  doublet droplets can be resolved with this method. cDNA preparations were performed according to 10x Genomics recommendations, amplified for 10–12 cycles and verified on fragment analyser. Both cell multiplexing oligo, and gene expression libraries were sequenced on a Novaseq (Illumina protocol #1000000106351 v03) with the cbot2 chemistry.