Sheep embryos were digested in calc-Mg-free HBSS containing collagenase Ⅱ (Thermo Fisher Scientific, 17101015, 100 U/ul) and collagenase Ⅳ (Thermo Fisher Scientific,17104019,100 U/ul) at 37℃, and the optimal digestion time was determined to be 15 min through pre-experiment. After digestion for 15 min, the dissociated cells were passed through a 70μm filter, centrifuged at 400g at 4℃ for 5 min. After the supernatant was removed, the pelleted cells were suspended in red blood cell lysis buffer (Miltenyi) and incubated on ice for 5 min to lyse red blood cells. After cracking red, the reaction was terminated with PBS+2%FBS. Centrifuged at 400g at 4℃ for 5 min. Then discard the supernatant and re-suspend, centrifuged at 300g at 4℃ for 5 min, discard the supernatant and re-suspend, the dissociated cells were passed through a 40μm filter element, and counted by Countstar automatic counter (Countstar Rigel S5). Approximately 17,000 cells were loaded into the 10x Chromium Controller. Library preparation was performed using the Single-Cell 3′ Reagent Kit v3.1 following the manufacturer's protocol. GEM-Reverse Transcription (GEM-RT) was carried out in a thermal cycler under the following conditions: 53°C for 45 minutes, followed by 85°C for 5 minutes, and finally held at 4°C.  After reverse transcription and cell barcoding, emulsions were broken, and cDNA was isolated and purified using a Cleanup Mix containing DynaBeads and SPRIselect reagent (Thermo Fisher Scientific). The purified cDNA was subsequently amplified by PCR. cDNA quality was assessed using the Agilent Bioanalyzer. For library construction, amplified cDNA was fragmented and end-repaired, followed by double-sided size selection. The library was then PCR-amplified with sample indexing primers and subjected to an additional double-sided size selection step.  The final libraries were purified and quality assessed according to the manufacturer's guidelines.