Single-cell RNA sequencing of adult rat testes

Here we present single-cell RNA sequencing data from10,983 adult rat testicular cells treated with ethanedimethyl sulphonate (EDS), which temporarily eliminates Leydig cells. The elimination and recovery of Leydig cells represented a complete testosterone depletion and restoration circle. The data-set includes all developing germ cells from spermatogonia to spermatozoa and somatic Sertoli and Leydig cells and is ideal for characterizing all developmental germ cells, their regulatory networks, and novel stage-specific markers. The data-set is particularly useful in exploring the effects of the androgen environment on regulation of spermatogenesis. The data-set contains 4 samples of single cell RNA sequence of whole rat testicular cells. Each sample contains 2 raw data files in .fastq format. There are 8 data files for the whole data-set. Sample 1 (C): from 3 untreated control rats. Data file names: C_1.fq.gz; C_2.fq.gz Sample 2 (E1W): from 3 rats 1 week after EDS treatment. Data file names: E1W_1.fq.gz; E1W_2.fq.gz Sample 3 (E3W); from 3 rats 3 weeks after EDS treatment. Data file names: E3W_1.fq.gz; E3W_2.fq.gz Sample 4 (E7W): from 3 rats 7 weeks after EDS treatment. Data file names: E7W_1.fq.gz; E7W_2.fq.gz The datasets were generated by 3 steps: 1) EDS treatment of adult SD rats To eliminate Leydig cells from the testes, nine rats were injected with a single dose of ethanedimethyl sulphonate (EDS; i.p., 80 mg/kg of BW) dissolved in a mixture of DMSO:PBS (1:3) solvent. Three rats were treated with DMSO:PBS vehicle as controls. Testes from 3 animals were collected at 1, 3, or 7 weeks post-EDS treatment, denoted as E1W, E3W or E7W, respectively, or 7 weeks post vehicle treatment, denoted as C for control. The treatment protocol insured complete Leydig cell elimination by 1 week and partial or complete Leydig cell regeneration by 3 or 7 weeks, respectively. The treatments were arranged in such a way that all animals were ready for tissue collection on a same day. Serum testosterone concentrations were assayed using the Immulite 2000 Total Testosterone Assay Kit (Siemens, Germany) with a detection sensitivity of 0.15 ng/ml and intra-assay coefficient of variation of 8.3%. 2) Preparation of testicular cell suspensions To eliminate possible contamination from blood cells, the testicular artery was cannulated and perfused with culture medium M199 containing 2.2 g/l Hepes, 0.1% BSA, 0.7 g/l sodium bicarbonate, pH 7.4) to clean out blood19. After decapsulation, the testes from 3 animals were pooled and then digested with 1 mg/ml collagenase-IV in DMEM/F12 medium at 34°C for 30 min with slow shaking (90 cycles/min) as described previously19. After allowing the undigested tissues to settle for 30 seconds, the dispersed cells in supernatants were filtrated through 2 layers (100µm pore on top of 30µm pore) of nylon mesh and washed twice with PBS. Cell viability was assayed by 0.4% Trypan blue exclusion and was consistently above 85% for all 4 groups. 3) Single-cell transcriptomes sequenced by 10X Genomics Chromium Cell capture, 10x Genomics library preparation, and sequencing were done by Novogene (Beijing, China). After washing twice in PBS, ~7000 cells were loaded onto 10x Chromium chips with 3’v2 chemistry and barcode to achieve a targeted cell count of 4000, according to the manufacturer’s instructions (10x Genomics, Pleasanton, CA). After cDNA synthesis, 14 amplification cycles were carried out for each library preparation. The resultant libraries were sequenced using 2 × 150 paired-end sequencing protocol on an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA), with a read length of 26 bp for cell barcode and unique molecule identifier (UMI) (read 1), 8 bp i7 index read (sample barcode), and 98 bp for actual RNA read (read 2). Each treatment group yielded approximately 550M reads. All downstream single-cell analyses were performed using Cell Ranger and Seurat software.