GSM8819787: Murine epididymal sperm, freshly isolated from three 6-month-old C57BL/6NHsd mice; Mus musculus; RNA-Seq
Total RNA was extracted from murine epididymal spermatozoa isolated from three 6-month-old C57BL/6NHsd male mice. Sperm population was purified by gradient centrifugation using ISolateTM (Irvine Scientific, CA) density gradient. 80% and 40% ISolateTM gradients were prepared with HS solution. Sperm purification was carried out by overlaying epidydimal sperm suspension on top of 40% gradient, and centrifuged for 30 min at 300g. Pure sperm cells were collected at the bottom and visually examined for purity and contamination under phase-contrast microscope. Only 100% pure sperm samples were used for the RNA extraction. To exclude potential contamination with leukocytes, comparison of both transcriptomes was used to determine sperm specific transcripts. Peripheral blood leukocytes (Pbl) were obtained from three 6-month-old C57BL/6NHsd male mice postmortem using heart puncture. Total amount of blood collected was 2 mL. Total RNA was extracted from cells using Qiagen RNeasy kit, and converted to cDNA followed by fragmentation to 200 bp. Total RNA obtained from murine Pbl was 55.2 µg and total RNA obtained from murine sperm was 14.73 µg; both RNA isolations have purity above A260/A280 = 2,1. DNA fragments with appropriate lengths (e.g., 200 bp) were purified using gel electrophoresis and sequencing libraries were prepared with poly(A)+-enriched RNA using Illumina mRNA-Seq Sample Prep Kit according to the manufacturer's instructions. Illumina Single Read Cluster Generation Kit v2 (Lot 4941271) was used for cluster generation. Final libraries concentrations were: mouse sperm: 49.1 ng/mcL; mouse leukocytes: 41.4 ng/mcL. The final libraries were assessed using Bioanalyzer to check fragment size, concentration, and purity. Sperm library (well #4) and pbl library (well #5) were loaded onto a single-read flow cell, where they underwent bridge amplification to form clonal clusters. Libraries were sequenced on the Illumina Genome Analyzer II using a 36-cycle Run v3 sequencing kit (Lot 4935209) by standard protocols. Sequences were aligned to mouse genome GRCm39. Bioinformatics support was provided by Centrillion Biosciences Inc. (Palo Alto, CA).