title: GSM7675226: Human_DMSO_1_6; Homo sapiens; RNA-Seq
library_construction_protocol: Human and mouse embryos were subject to immunosurgery to remove outer trophectoderm cells and isolate the inner cell mass (ICM), as reported previously(Strawbridge et al., 2002: PMID 34870826;Solter and Knowles 1975 PMID 1108013). The zona pellucida was removed by washing embryos through drops of acid Tyrode's solution pre-warmed to 37°C. Embryos were then washed briefly and incubated at 37°C for 30min in 20% anti-mouse serum (Sigma) or 20% anti-human serum (Sigma), depending on the species, in Global media + 10% HSA. Embryos were then washed briefly in Global medium + 10% HSA, before incubation with 20% Guinea Pig Serum Complement (Merk) at 37°C for 10-15min until trophectoderm began to lyse. Embryos were then moved to a drop of medium and triturated with 100µm STRIPPER tip (Cooper Surgical) to remove lysed trophectoderm and isolate the inner cell mass.Isolated ICMs were then briefly washed, then incubated at 37°C for 3 min in 0.5% Trypsin, 1mM EDTA in PBS. Then, ICMs were transferred to 4% BSA, 0.5mM EDTA in PBS for fine manual dissociation into single cells with pulled (Sutter Instruments) glass capillaries (World Precision Instruments). Cells were then picked manually with pulled glass capillaries for downstream single-cell RNA-seq. cDNA kit lot number 2109360A Single-cell cDNA synthesis was performed using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara) according to the manufacturers protocol with some modifications as previously described(Blakeley et al., PMID 26293300). Briefly, single-cells were manually picked after dissociation and snap frozen on dry ice in 5µl 10x Reaction Buffer in low-bind 0.2ml PCR tubes and stored at -80°C until processing. Samples were subject to first strand cDNA synthesis, then cDNA was amplified by LD PCR for 23 cycles. Amplified cDNA was purified on AMPure XP beads, using a 96-well magnetic stand (ThermoFisher), and eluted in 17µl of elution buffer. Single-cell RNA-seq libraries were prepared using a Nextera XT DNA Library Preparation Kit (Illumina) using according to the manufacturers protocol. Sequencing was carried out as paired-end 50bp read on Novaseq 6000 to a depth of 12 million reads.