GSM5949216: Adult sample 1; Callithrix jacchus; RNA-Seq
Testicular tissues were obtained from neonatal (n=3; 1-2 days old), pre-pubertal (n=3; 6 months old) and adult (n=2; 4, 10 years) marmoset monkeys (Callithrix jacchus). Single cell suspensions were obtained using a two-step enzymatic digestion (Kossack et al., 2013; Laurentino et al., 2019). A total of 20 000 cells were used for ploidy analysis, 12 000 cells were prepared for scRNA-seq. Ploidy analysis was performed on 20 000 cells of each sample prior to scRNA-seq. After centrifugation, the cells were incubated in the dark for 30 minutes with a solution containing 50 mg/ml Propidium Iodide (Sigma- Aldrich, Cat# P4170), 1 mg/ml bovine serum albumin (Sigma-Aldrich, Cat# A9647), 0.1% Triton X-100 (Sigma-Aldrich, Cat# 93443), and 10 mg/ml RNase A (Sigma-Aldrich, Cat#R6513) in phosphate-buffered saline. After incubation, approximately 10 000 cells were analyzed for each sample using a Beckman Coulter CytExpert QC Flow Cytometer. Debris was defined based on forward and side scatter and excluded from the analysis. The cells analyzed for DNA content were measured at 617 nm and assigned to the following categories according to staining intensity: 1) haploid cells (1C: spermatids), 2) diploid cells (2C: spermatogonia, somatic cells (e.g., Leydig peritubular and Sertoli cells)), 3) ''double diploid'' cells (4C: spermatocytes). 12 000 cells per sample (neonatal (n=3), pre-pubertal (n=3), adult (n=2) were suspended in MEMa at a concentration of 500 cells/ml and loaded onto the Chromium Single Cell A Chip. Library preparation was performed following the kit instructions (Chromium Single Cell Kit [v2, v3 chemistry]). Briefly, 6 000 cells per sample were captured by the 10x Genomics Chromium controller, after cDNA synthesis, 12-14 cycles were used for library amplification. The resulting libraries were quantified using the Lab901 TapeStation system (Agilent) before shallow sequencing (NextSeq 550 sequencer, Illumina) for quality control. The final, deep sequencing was performed on a NovaSeq 6000 sequencer (Illumina), using 2x150 bp paired-end sequencing.