title: GSM6745908: 19R018_zygotene spermatocytes-Dhx36-cKO-rep2; Mus musculus; RNA-Seq
library_construction_protocol: Testes from from wild-type and Dhx36 mutant spermatogenesis synchronous mice were collected in PBS on ice. After removal of the tunica albuginea, the testes were incubated in 5mL of PBS with 120 U/mL of collagenase type I (Worthington) at 32 °C with gentle agitation for 5 min. The dispersed seminiferous tubules were then digested with 5mL of 0.25% trypsin (Gibco), 0.1 mL of DNase I (5 mg/mL; Sigma) at 32 °C for 8 min, and then terminated by adding 0.5 mL of fetal bovine serum (FBS; Gibco) to inactivate trypsin. The resulting dissociated testicular cells were filtered through a PBS-prewetted cellular filter (70 μm). After spin down at 500 g for 5 min at 4 °C, the cells in the pellet were resuspended at a concentration of 106 cells/mL in DMEM with Hoechst 33342 (3 mg/mL; Sigma) and 5 μl DNase I followed by rotating for 20 min at 32 °C in the oven at 10 r.p.m./min speed. Zygotene spermatocytes were collected based on their fluorescent label with Hoechst 33342 staining using FACS (BD Biosciences). Cell purity was further examined by meiotic nuclear spreading with immunofluorescence staining as described above. The purity of isolated zygotene is about 95% Total RNA was extracted from zygotene spermatocytes isolated from wild-type and Dhx36 mutant spermatogenesis synchronous mice using Trizol reagent (Invitrogen). RNA quality was determined by Qubit Fluorometer and Agilent Bioanalyzer 2100. rRNAs were removed from total RNAs by the NEBNext rRNA Depletion Kit (NEB). The remaining RNAs were fragmented and then reverse transcribed. Libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) according to the manufacturer's instructions.