GSM8461057: Morphine, 90 days abstinent _ BIO REPLICATE 4; Rattus norvegicus; miRNA-Seq
Sperm cells extracted from cauda epididymus and purified using a double swim_up assay. For sperm RNA extraction: Sperm cells were resuspended in nucleus isolation buffer (15mM Tris_HCL, pH 7.5, 60mM KCl,15mM NaCl, 5mM MgCl2, 1mM CaCl2, 250mM sucrose, 1mM dithiothreitol). Trizol_LS solution was added, and samples were homogenized using a Dounce homogenizer, then run through a 27g needle. Lysate was spun down to pellet salts and debris. Supernatant then combined with chloroform and spun down. Aqueous phase was combined with isopropyl alcohol & glycogen and incubated overnight at _20C to precipitate RNA. RNA pellets were washed twice with cold 75% EtOH. The final RNA pellet was resuspended in RNAse_free water. Small RNA libraries were prepared from sperm RNA using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina. Libraries were purified using the Qiaquick PCR purification kit, then size selected using AMPure XP beads.