GSM7635168: Sperm cell sRNA-Seq - VSL11; Homo sapiens; miRNA-Seq
Total RNA from semen samples was extracted using the Maxwell® RSC miRNA Tissue (Promega). Initially, 200ul of sperm samples were centrifuged for 15 minutes at 12,000x g at 4°C, discarded the supernatant and added at the pellet 200ul of Phosphate buffered saline (PBS), after centrifuge for 30 minutes at 5,000g at 4° C. At the end of the automated extraction, RNA samples were eluted in 50ul, following the manufacturer's instructions. Small RNA-seq libraries were prepared using the NEBNext® Multiplex Small RNA Library Prep for Illumina® (New England Biolabs, Inc.) kit used to convert small RNA transcripts into barcoded cDNA libraries. For each library, 200 ng of RNA were processed as starting material. Each library was prepared with a unique indexed primer. Multiplex adapter ligations, reverse transcription primer hybridization, reverse transcription reaction and PCR amplification were performed according to the manufacturer's protocol. After PCR amplification, the cDNA constructs were purified with the QIAQuick® PCR Purification Kit (QIAGEN), following the modifications suggested by the NEBNext® Multiplex Small RNA Library Prep for Illumina® protocol. Final libraries were loaded on the Bioanalyzer® 2100 (Agilent Technologies) using the DNA High Sensitivity Kit (Agilent Technologies) according to the manufacturer's protocol. Libraries were pooled together (34-plex) and further purified with a gel size selection. A final Bioanalyzer® 2100 run with the High Sensitivity DNA Kit (Agilent Technologies) allowed the assessment of DNA libraries quality regarding size, purity, and concentration. The obtained libraries were subjected to the Illumina sequencing pipeline on Illumina NextSeq500 sequencers (75 cycles, Illumina Inc., USA).