A probability of stealth multiplets in the sample-multiplexed scRNA-seq
A probability of stealth multiplets in the sample-multiplexed scRNA-seq
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 DMEM+serum/LIF, including passages 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, add 150ul N2B27+3uM CHIR99 (Wnt agonist). The distal region of a hind limb bud were isolated from a mouse embryo at E11.5. The tissue was loosened with TrypLE Express (Thermo Scientific), and the ectoderm was removed. The isolated mesenchyme cells were further dissociated by pipetting. NIH3T3 cells were cultured with DMEM, supplemented with 10% Fetal bovine serum, L-glutamine, and Penicillin/Streptomycin. MULTI-seq was performed as described in the original article (McGinnis et al. Nat Method 16:619-626, 2019). cDNA and MULTI-seq barcodes are fractionated with SPIRselect (Beckman Coulter) after the cDNA amplification step. A cDNA library was constructed with Chromium Single Cell 3' GEM, Library and Gel Bead Kit v2 (10x Genomics) according to the manufacturer's protocol. MULTI-seq barcodes were amplified with KAPA HiFi HotStart ReadyMix (Roche) and single-indexed with the TruSeq-RPI primers for sequencing along with the cDNA library.