title: GSM7025381: urine_TESEN_23; Homo sapiens; miRNA-Seq
library_construction_protocol: miRNA extraction from seminal plasma was performed using the Qiagen RNeasy Mini Kit (74104; Qiagen, Antwerp, Belgium). The samples were thawed and resuspended. First, 500 µL Trizol was added to 400 µL seminal plasma and incubated for 5 minutes at RT. Next, 150 µL chloroform (102445; Merck) was added and the samples incubated for 3 minutes at RT. The sample was centrifuged at 11,900g for 15 minutes at 4°C. The aqueous phase was transferred into a new tube and one volume of 70% ethanol was added. Up to 700 µL of the sample was transferred to a RNeasy spin column in a 2 mL-collection tube and centrifuged for 15 seconds at >8000g at 4°C. The flow-through was discarded and the previous step was repeated until the whole sample was transferred to the spin column. Then, 700 µL buffer RWT was added to the spin column and centrifuged (15 seconds; >8000 x g ; 4°C), after which the flow-through was discarded. A total of 500 µL RPE buffer was added in the same manner. Next, 500 µL 80% ethanol was added and the sample was centrifuged for 2 minutes at >8,000g at 4°C. The collection tube with flow-through was discarded and a new 2 mL-collection tube was placed under the spin column. This was centrifuged with open lid at full speed for 5 minutes. Finally, the column was placed on a 1.5 mL-collection tube, 20 µL RNase-free water was added and the sample was centrifuged for 1 minute at >8000g at 4°C to elute the RNA.   miRNA extraction from the urine samples was performed using the Norgen Urine miRNA Purification Kit (29000; Norgen Biotek Corp., Thorold, Canada), according to the manufacturer's instructions with a minor adaptation to the protocol (usage of 50 µL elution solution A instead of 20 µL). Sequencing libraries were generated from 50 ng RNA input material using the SMARTer smRNA-Seq Kit for Illumina (Takara) according the instructions of the manufacturer. Size selection of final libraries was done using AMPure Xp beads. After library quality and fragmentation control on fragment analyzer (Agilent) samples were pooled and sequenced on a Novaseq 6000 platform (Illumina) using paired end sequencing (100*8*8*100).