A dataset of scRNA-seq for rat testicular interstital cells without Leydig cells

The data-set contains 3 samples of single cell RNA sequence of rat testicular interstitial cells. Three samples cover Leydig cell elimination and partial recovery. The data-set is useful in studying stem Leydig cell characteristics and differentiation. Each sample contains 2 raw data files in .fastq format. There are 6 data files for the whole data-set.  Sample 1 (C2a): from 3 untreated control rats. Data file names: C2a_1.fq.gz; C2a_2.fq.gz Sample 2 (E1W2a): from 3 rats 1 week after EDS treatment. Data file names: E1W2a_1.fq.gz; E1W2a_2.fq.gz Sample 3 (E3W2a); from 3 rats 3 weeks after EDS treatment. Data file names: E3W2a_1.fq.gz; E3W2a_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 E1W2a or E3W2a, respectively, or 3 weeks post vehicle treatment, denoted as C2a 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 blood. After decapsulation, the interstitial fractions were dissected from testes of 3 animals and 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 previously. 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 3 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, ~20,000 cells were loaded onto 10x Chromium chips with 3’v2 chemistry and barcode to achieve a targeted cell count of 10,000, 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.

该数据集包含3份大鼠睾丸间质细胞的单细胞RNA测序样本。这3份样本涵盖了莱迪细胞清除及部分恢复的过程。该数据集对于研究干细胞莱迪细胞的特征和分化具有重要意义。每个样本包含2个原始数据文件，格式为.fastq。整个数据集共包含6个数据文件。样本1（C2a）：来自3只未经处理的对照大鼠。数据文件名：C2a_1.fq.gz；C2a_2.fq.gz；样本2（E1W2a）：来自3只接受EDS治疗1周后的大鼠。数据文件名：E1W2a_1.fq.gz；E1W2a_2.fq.gz；样本3（E3W2a）：来自3只接受EDS治疗3周后的大鼠。数据文件名：E3W2a_1.fq.gz；E3W2a_2.fq.gz。数据集的生成经过以下3个步骤：1）对成年SD大鼠进行EDS处理，以消除睾丸中的莱迪细胞，9只大鼠通过皮下注射单剂量的乙撑亚磺酸（EDS；剂量为体重80 mg/kg，溶解于DMSO:PBS溶剂中，比例为1:3）进行处理。3只大鼠作为对照组接受DMSO:PBS溶剂。在EDS处理后1周、3周或7周，分别收集3只动物睾丸，分别标记为E1W2a或E3W2a，或车辆处理后3周的C2a。治疗方案确保在1周内完全消除莱迪细胞，并在3周或7周内部分或完全再生莱迪细胞。所有动物均安排在同一天进行组织收集。使用Siemens公司（德国）的Immulite 2000总睾酮测定试剂盒测定血清睾酮浓度，检测灵敏度为0.15 ng/ml，试验内变异系数为8.3%。2）制备睾丸细胞悬液，为消除血液细胞的潜在污染，对睾丸动脉进行插管，并用含有2.2 g/l Hepes、0.1% BSA、0.7 g/l 碳酸氢钠，pH 7.4的培养基M199进行灌注，以清除血液。去包膜后，从3只动物的睾丸中分离出间质部分，并混合后用1 mg/ml的胶原酶-IV在DMEM/F12培养基中于34°C消化30分钟，缓慢摇动（90次/分钟）。在允许未消化的组织沉降30秒后，通过2层（上层100µm孔径，下层30µm孔径）尼龙网过滤分散的细胞，并使用PBS洗涤两次。通过0.4%台盼蓝排斥法检测细胞活力，所有3组均保持在85%以上。3）利用10X Genomics Chromium进行单细胞转录组测序，细胞捕获、10x Genomics文库制备和测序由北京诺维基因科技有限公司完成。在用PBS洗涤两次后，约20,000个细胞被加载到带有3’v2化学和条形码的10x Chromium芯片上，以实现10,000个目标细胞计数，根据制造商的说明进行（10x Genomics，Pleasanton，CA）。在cDNA合成后，每个文库进行14次扩增循环。使用Illumina NovaSeq 6000平台（Illumina，San Diego，CA）上的2 × 150端到端测序方案进行测序，细胞条形码和唯一分子标识符（UMI）（读1）的读长为26 bp，8 bp i7索引读（样本条形码），实际RNA读（读2）为98 bp。每个处理组产生约550M reads。所有下游的单细胞分析均使用Cell Ranger和Seurat软件进行。