GSM9787066: Cryopreserved sperm - replicate 4; Homo sapiens; miRNA-Seq
Total RNA was extracted from 200 μL seminal plasma or from sperm pellets using TRIzol LS Reagent (Invitrogen, USA) or TRIzol Reagent (Invitrogen, USA), respectively, following the manufacturer's protocols. Briefly, samples were mixed with TRIzol reagent and incubated at room temperature for 5 min. Chloroform was added, and the mixture was centrifuged at 12,000 × g for 15 min at 4°C. The aqueous phase was collected, and RNA was precipitated with isopropanol. The RNA pellet was washed with 75% ethanol, air-dried, and dissolved in RNase-free water. RNA concentration and purity were assessed using a NanoDrop ND-2000 spectrophotometer (Thermo Fisher Scientific, USA). RNA integrity was evaluated using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) with the RNA 6000 Pico Kit. Small RNA libraries were constructed using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (New England Biolabs, USA) following the manufacturer's protocol. Briefly, 3' and 5' adapters were sequentially ligated to the small RNA fraction. Reverse transcription was performed to generate first-strand cDNA, followed by PCR amplification (15 cycles). The PCR products were purified by PAGE gel electrophoresis to select fragments corresponding to small RNA insert sizes (140-160 bp). Library quality and concentration were assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA) with the High Sensitivity DNA Kit.