GSM8676527: Round spermatids, Ago413+/+ testes, rep2, RNA-seq; Mus musculus; RNA-Seq
Single cell suspension from testes was obtained following the method described by Ascenção et al.Testes from 2-5 mice were collected, the tunica albuginea was removed, and tubules were placed in 10 ml of preheated (37°C) DMEM-F12 containing 2 mg of Collagenase 1A (Sigma) for 2 minutes with manual shaking. Digestion was stopped by two washes of the tubules with DMEM-F12. Next, the tubules were incubated in 10 ml of DMEM-F12 with 5 mg of trypsin (Sigma) and 7 mg/ml of DNAse I (Sigma) at 37°C with constant shaking (150 rpm) until the length of the tubules was about 1 mm (approximately 10 minutes). Digestion was stopped by addition of 3 mls of Fetal Bovine Serum (FBS, Sigma) and 30 ml of DMEM F-12. Digested tubules were strained on a 100 μm strainer and centrifuged for at 15°C for 5 minutes at 600g. The single cell suspension of germ cells obtained was then resuspended in FBS and enriched fractions of pachytene spermatocytes and round spermatids were obtained using the gravity-density BSA gradient method MDR developed by Da Ros and others. After collecting the corresponding fractions, cells were pelleted and washed in cold PBS by centrifugation. Purity of each fraction was evaluated by light microscopy and immunofluorescence using the marker SYCP3 and DAPI, and only fractions containing above 70% pachytene spermatocytes and round spermatids were pooled. Total RNA was isolated with Trizol, with an extra chloroform extraction to remove residual phenol and addition of glyco-blue as a carrier to promote RNA precipitation. RNA concentration was determined with a Qubit and integrity assessed on a Fragment Analyzer. [Directional] RNA-seq libraries were prepared from 100-200ng total RNA using the NEBNext Directional Ultra II RNA Library Prep Kit for Illumina (New England Biolabs), with initial polyA+ isolation.