GSM9135578: E13.5_placenta_yolk sac_MO; Mus musculus; RNA-Seq
Pregnant mice were euthanized by CO₂ asphyxiation followed by cervical dislocation. Embryonic tissues, including placenta, yolk sac, and fetal liver at embryonic day 13.5 (E13.5), and fetal liver at E17.5, were collected. For each tissue type, nine embryos per treatment group—control (CT) and maternal obesity (MO)—were harvested. Tissues from each group were pooled and placed into 50 mL conical tubes containing DMEM supplemented with 4% fetal bovine serum (FBS), and kept on ice to preserve cell viability.  Tissues were minced into approximately 1 mm fragments using fine scissors under sterile conditions in a biosafety cabinet. The tissue fragments were then digested in 10 volumes of DMEM containing 2.5% FBS, 100 μg/mL Collagenase Type IV, 50 μg/mL DNase I, and 100 μg/mL heparin. To preserve erythroid cells, red blood cell lysis was not performed. Digestion was carried out at 37 °C in a humidified incubator with 5% CO₂ for 30 minutes.  The enzymatic reaction was stopped by adding an equal volume of DMEM containing 2.5% FBS. The cell suspension was centrifuged at 800 rpm for 5 minutes at room temperature. After discarding the supernatant, the cell pellet was resuspended in PBS supplemented with 0.4% FBS and filtered through a 40 μm cell strainer to remove debris and aggregates.  Cell concentration and viability were assessed using trypan blue exclusion and a hemocytometer. Only samples with viability greater than 80% were used for downstream single-cell RNA sequencing. Final cell suspensions were adjusted to a concentration of 1000 cells/μL. Single-cell RNA-seq libraries were prepared using the 10x Genomics Chromium GEM-X Single Cell 3' v4 Reagent Kit, following the manufacturer's standard protocol. Libraries were sequenced on an Illumina NextSeq 2000 platform with paired-end reads.