title: GSM7351781: TOPBP1B5/B5 testes,rep2,scRNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: Mice testis were collected from 8-12 weeks old mice (n = 5 mouse, 10 testis for Topbp1+/+ , and  n = 20-30 mice, 40-60 testis for  Topbp1B5/B5). Decapsulated testes were held in 10 ml of DMEM-F12 buffer containing 2mg of Collagenase 1A and DNAse 7 mg/ml at 35°C in a shaker water-bath at 150 rpm for 5 min. 40 ml of DMEM-F12 was added and the tubules were left to settle for 1 min. The supernatant was removed and tubules were washed again with 40 ml of DMEM-F12. The tubules were digested using 10 ml of DMEM-F12 buffer containing 5 mg of trypsin in a 35°C shaker water-bath, 150 rpm for 5 min. Digested tubules were strained on a 100 μm strainer containing 3 ml of FBS. Cells were centrifuged at 300xg for 5 min, 4°C and checked for single cells and viability. Cells were stained with Vybrant dye cycle 100 μM for 30 min at room temperature. After staining, cells were sorted to enrich for spermatocytes using the Sony MA900 sorter, tuned to emit at 488 nm and with a 100 μm nozzle. Laser power was set to collect VDG-emitted fluorescence in FL1. Sorted cells were collected on 1.5 ml tubes containing 0.5ml of DMEM-F12 buffer + 10% FBS. 10X Genomics Chromium Single Cell 3′ RNA-seq v2