Illumina NovaSeq 6000 paired end sequencing; Marmoset epiblast and amnion spheroids as models for postimplantation lineage specification
cmPSC were encapsulated in agarose microgels and transferred into conventional tissue culture plates with or without mouse embryonic fibroblasts (MEFs) in differentiation permissive, serum-free N2B27 supplemented with 1% Matrigel, 2.5% Chemically Defined Lipids (Gibco) and 1% Penicillin-Streptomycin. Signalling inhibitors and activators were then added to this base medium: FGF (bFGF, 100 ng/ml, Cambridge Stem Cell Institute), CHIR (CHIR99021, 3 µM, Cambridge Stem Cell Institute),  XAV (XAV939, 3 µM,  Cell Guidance Systems SM38-200),  ACTA (Activin A, 20 ng/ml,  Cambridge Stem Cell Institute),  SB43 (SB431542, 10 µM, Tocris 1614),  BMP4 (BMP4, 20 ng/ml, R&D 314-BP).  For each condition, a representative pool of 20-30 microgels were randomly selected for profiling by single-cell RNA sequencing. Structures were released from microgels by incubation with 1 U/ml Agarase (Thermo Fisher Scientific, EO0461) and dissociated for 10-15 minutes in 0.25% Trypsin-EDTA (Gibco, 25200056) followed by manual dissociation in N2B27 using a blunted microcapillary pipette. Single cells were isolated manually using a microcapillary pipette, transferred immediately into 4 µl single cell lysis buffer and snap frozen. RNA separation was performed using biotinylated oligo-dT30VN-tailed oligonucleotides (IDT) conjugated to Dynabeads Streptavidin C1 in an RNAse-inhibitor supplemented buffer solution.  Sequencing libraries were prepared according to an adapted version of the G&T protocol previously described. Samples were transferred to a 96-well plate and prepared for both transcriptome and methylome sequencing, by physical separation of genomic DNA (gDNA) from messenger RNA (mRNA).