GSM4839042: 063-mv4-Control; Homo sapiens; miRNA-Seq
Total RNA including sncRNA was extracted from frozen sperm pellets using the RNeasy Micro Kit (Qiagen, UK) with a modified protocol adapted from Goodrich, et al., 2012 which was optimised for recovery of sperm RNA. Briefly, sperm pellets were thawed, mixed with Qiazol (Qiagen, UK) and manually disrupted. Chloroform was added, samples incubated (room temperature for 5 minutes) and centrifuged (12000 x g) for 20 minutes. The upper aqueous layer was removed and mixed with ethanol. Samples were added to micro RNeasy spin columns (Qiagen, UK) and centrifuged for 15 seconds (8000 x g). Spin columns were washed using supplied buffers and ethanol. Samples were transferred to RNAse free collection tubes and total RNA eluted. RNA concentrations were quantified using Qubit total and microRNA assays (Thermo Fisher, UK), RNA was snap frozen on dry ice and stored at -80 degrees. Sperm RNA samples were selected from both control and dietary intervention groups based on the quantity of RNA post extraction (n=17). 10µl of RNA was used to perform small RNA-seq (~15 million reads) using the smallRNA workflow (Oxford Genomics, OGC). NEBNext Small RNA Library Prep Set for Illumina (E7330) was used as per manufacturers' instructions. Briefly, 3' SR adapter ligation was carried out followed by RT primer hybridisation to the mRNA. Denatured 5' SR adapter was then ligated, and first strand cDNA reverse transcription was performed. After second strand synthesis with indexed (TruSeq-based single indexed i7) and P5 primers, dsDNA was then cleaned-up using Ampure XP beads, and the libraries were size selected by Pippin (125-160bp). The concentration of the small RNA libraries was determined by Picogreen before multiplexing for sequencing. The final size distribution of the pooled library was determined using Agilent Tapestation, and quantified by Qubit (Thermofisher, UK), before sequencing on the HiSeq2500 (Illumina, UK) (2X runs) as 50bp single reads. Data were aligned to the reference genome and FASTQ files generated.