GSM5328072: AMC-72-post; Mus musculus; RNA-Seq
Total RNA was isolated from frozen mature sperm from adult male mice using a protocol combining TRIzol/chloroform extraction with RNeasy MiniElute Cleanup kit. In short, 200 µl Trizol was used to homogenize the tissue. An additional 500 µl Trizol was added along with 140 µl chloroform. The lysate was centrifuged at 12,000xg for 15 minutes and the aqueous layer was removed and added to the RNeasy mini spin column. The rest of the protocol was completed as outlined by the manufacturer. Samples were treated with DNAse to rid samples of genomic DNA. After extraction, purity and concentration were measured on an Agilent 2100 Bioanalyzer (Agilent, Santa Clara CA) using an RNA 6000 Nano assay. Libraries were prepared using the TruSeq Stranded Total RNA Library Prep Gold protocol (Illumina, San Diego, CA). Briefly, cDNA was synthesized from ribo-depleted RNA, followed by end-repair and ligation with sequencing adaptors. The libraries were then size selected and purified using AMPure XP beads (Beckman Coulter, Brea, CA). Barcode bases (8 bp) were introduced at one end of the adaptors during PCR amplification steps. Library size and concentration were measured by Bioanalyzer or Tape Station (Life Technologies, Grand Island, NY) before sequencing. All libraries were pooled for multiplexing and sequenced on the Illumina HiSeq 2500 System utilizing V3 chemistry with 150 base pair double-end reads at the sequenced on a HighSeq2500 System using V4 chemistry with 50 base pair single-end reads at GeneWiz LLC (South Plainfield, NJ).