Accutase medium

Selected oocytes or embryos were transferred to acidic Tyrode’s solution (pH 2.5, Sigma) to remove the zona pellucida. Zona-free embryos were then incubated for 10 min (for the 2-, 4-, and 8-cell stage embryos) or 20 min (for morulae) in Accutase medium (Gibco) before dissociation into single embryo cells by careful pipetting. We then manually separated blastocysts into ICM and TE using laser microdissection. ICM and TE compartments were subsequently placed into Accutase medium for 30 min and washed thoroughly in phosphate-buffered saline (PBS) with 0.5% (m/v) bovine serum albumin (BSA) (Sigma). We isolated single embryo cells by gentle, repeated pipetting; washed them 3–5 times in PBS with 0.5% BSA; and placed the cells into a PCR tube for LiCAT-seq library preparation.

Both ST blastocysts and ICSI control blastocysts were collected on days 5 to 7 postfertilization, and no delayed or arrested embryos were included in this study. We first treated the blastocysts with acidic Tyrode’s solution (Sigma-Aldrich, Cat. T1788) using a mouth pipette to remove the zona. The blastocysts were then transferred to digestion medium (Accutase medium: 0.25% trypsin, 1:1) (Sigma-Aldrich, Cat. A6064; Gibco, Carlsbad, California, USA, Cat. 25300–056) drops at 37°C for 10 to 20 minutes with gentle and careful pipetting to isolate individual cells. The inner diameters of the glass pipettes varied from 10 to 30 μm. Individual cells were selected and collected into 200 μL PCR tubes with lysis media. All the procedures were performed under a stereomicroscope (Nikon, Shinagawa, Tokyo, Japan, Cat. SMZ745).