GSM8443344: Zfy12_1r; Mus musculus; RNA-Seq
About 20mg of frozen testis pieces were placed in Precellys Homogenizer tubes 2.8 mm ceramic reinforced (Precellys 50722019, Bertin Technologies, France) on dry ice. 500μl of Trizol reagent (Sigma-Aldrich, USA) was added to the tubes and the samples placed in a Bertin Precellys 24-Dual High-throughput Tissue Homogenizer (Bertin Technologies, France). The machine was then run with the factory programme 5: 6,500 rpm for 20 seconds, automatic 25 seconds of pause and a repeat of 6500 rpm for 20 seconds. The lysate was then centrifuged for 5 minutes at 12,000 x g at 4°C and the clear supernatant transferred to a MaXtract High Density phase lock tube (Qiagen, UK). After 5 minutes of incubation at room temperature, 100μl of chloroform was added and tubes shaken vigorously for 2-3 minutes. Tubes were spun down for 15 minutes at 12,000 x g at 4°C. The upper aqueous phase was transferred to a new tube and 250μl of isopropanol added and mixed by pipetting. After 10 minutes of incubation at room temperature samples were spun down for 10 minutes at 12,000 x g at 4°C. The supernatant was discarded, the RNA pellet washed with 1ml of 75% ethanol and the samples spun down for 5 minutes at 7,500 x g at 4°C. Finally, the pellet was air-dried for 5-10 minutes and resuspended in 40μl of nuclease-free water. Libraries were prepared using the NEBNext Ultra II Directional PolyA mRNA kits according to manufacturer instructions.