GSM8141452: Stage 21F, scRNA-seq; Trachemys scripta; RNA-Seq
At each developmental stage, 100 pairs of gonads underwent digestion for 20 minutes at 37°C in a 0.25% trypsin solution. Single-cell suspensions were filtered through a 40 μm cell strainer (431750, Corning) and washed two to three times with PBS. Cell viability was assessed using trypan blue staining with a TC20 automated cell counter (Bio-Rad, Hercules, CA). The viability of gonadal cells in the single-cell suspensions exceeded 85%. Single-cell suspension concentration was adjusted to between 700 and 1200 cells/μL. Cells from each sample were processed using Chromium Single Cell 3' Reagent Kits (v3 chemistry, CG000183) following the manufacturer's instructions at Novogene Bioinformatics Technology Co., Ltd., Tianjin, China. Input cells were loaded onto the Single Cell B Chip (v3 chemistry, PN-1000153) channels according to expected counts. 10x libraries were constructed using the Chromium Controller and Chromium Single Cell 3' Reagent Kits (v3 chemistry, CG000183). Briefly, single-cell suspensions were loaded into each channel of a Chromium Controller (10x Genomics, Pleasanton, CA) to generate single-cell gel beads in emulsion (GEMs). Subsequently, 10x libraries for each channel were prepared using the Chromium Single Cell 3' Gel Bead and Library Kit v3 (PN-1000153, 1000075, 120262; 10x Genomics). Sequencing was performed on an Illumina NovaSeq with 150-bp paired-end reads.