GSM3702694: Sample_116_longRNA; Homo sapiens; RNA-Seq
Sperm samples were brought up to 1ml volume with PureSperm Buffer (Nidacon) and laid onto a 50% PureSperm (Nidacon) gradient, then centrifuged at 300xg for 20 minutes. The pellet was removed and washed in PureSperm Buffer. Each sample was added to a 2ml tube containing 500ul RLT buffer (Qiagen) with 7.5ul BME (14.3M), 100mg 0.2mm nuclease free SS beads. The sample were homogenized, 500ul Qiazol (Qiagen) added and homogenized again. 200ul chloroform (0.124M) was added and samples shaken by hand then centrifuged at 12,000xg for 20 minutes at 4⁰C and the upper aqueous layer removed. RNA was isolated from the aqueous layer using a customized Qiacube (Qiagen) protocol. RNA samples were treated with 4U Turbo DNase (Ambion) for 20 minutes at room temperature.  Samples were tested for DNA contamination by RT-PCR and intron spanning PRM1 primers. Samples still containing DNA were treated a second time and tested again by RT-PCR. The RNA samples void of DNA were quantified with a fluorescent assay as described. An aliquot of RNA (5 ng or 10 ng) of each sample was used to make cDNA with SuperScript III RT (Invitrogen). The cDNAs were used as template for RT-PCR with intron spanning PRM1 primers providing verification of RNA. Two nanograms of RNA per sample was used with the Seqplex (Sigma-Aldrich) amplification kit prior to library construction. Fifty nanograms of Seqplex cDNA product was used with the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs) to create sequencing libraries. Complete barcoded libraries were quantified and pooled for sequencing at a concentration of 2 nM per sample. All samples were subject to paired-end sequencing using either the NextSeq 500 (Illumina) sequencer, HiSeq 2500 (Illumina) sequencer or the HiSeq 4000 (Illumina) sequencer. One nanogram of small RNA per sample was used with the NEXTflex Small RNA-Seq Kit v2 (Bioo Scientific) to create sequencing libraries. Complete barcoded libraries were quantified and pooled for sequencing. Samples were subject to paired-end sequencing using the MiSeq (Illumina) sequencer.