title: GSM2855106: DN0_1002; Homo sapiens; miRNA-Seq
library_construction_protocol: [sample collection and cell extraction] Blood was withdrawn between 8 and 9 AM after over-night fasting after excluding current infection and heavy physical activity, heavy meal, and alcohol consumption on the previous day. 3 x 9.5 ml blood was collected in EDTA monovettes (not containing heparin) for white blood cell isolation. Tubes were gently mixed by manually inverting ten times each. For plasma collection 2 x 8 ml were used; for white blood cell isolation 3 x 1.5 ml were further processed. Buffer EL (7.5 ml, Qiagen, cat#: 79217) was added and samples were incubated on ice for 25 min. At 7 and 15 min the samples were mixed and then centrifuged (400 g, 10 min, 4°C). The supernatant was discarded and the cell pellet was re-suspended in 3 ml of Buffer EL before centrifuged again (400 g, 10 min, 4°C). The supernatant was again discarded and the cell pellet re-suspended in 1.5 ml of RNA protect cell reagent (Qiagen, cat#: 76526). Samples were then stored at -80°C before further processing. Total RNA was extracted using the MiRNeasy miRNA isolation kit (Quiagen). All samples were kept on ice, tubes were centrifuged at 5000rcf to precipitate the cells, and overlaying RNA later solution was removed. Cells were disrupted with a plastic pestle and homogenized in 500µl (700µl for mouse tissues) Trizol lysis buffer. Following incubation for 5 min in room temperature 100 µl (accounting for 1:5 of the total volume) of chloroform was added to each sample. Samples were shortly vortexed and incubated for 2-3 min at room temperature, and were centrifuged at 4°C for 15 min at 12000rcf. The supernatant was mixed with absolute ethanol (1.25 times the supernatant volume) and the mixture was washed using the provided washing solutions and filter sets. Samples were eluted in 15µl (14µl for mouse tissues) RNAse free DDW. RNA quality and quantity was measured using a Nanodrop 2000 (PeqLab/Thermo Scientific). A total of 100µg per sample was run on an agarose gel for quality control. And RNA integrity was further tested using the 2100 Bioanalyzer (Agilent Technologies) using RNA 6000 Nano LabChip Kits (Agilent Technologies) and achieved an average RIN value of 7.5. An even amount of 150ng RNA per sample was used for small-RNA library construction using the New England BioLabs NEB-next multiplex small-RNA library construction kit. The library was size selected based on DNA 1000 LabChip kits by 2100 Bioanalyzer (Agilent Technologies) results to 146bp long transcripts which include the barcodes, adaptors and ca. 20bp long miRNAs. This ensures that sequenced transcripts are in the maximum length of 30 bp only. Libraries were quantified using the highly sensitive fluorescent dye-based Qubit® ds DNA HS Assay Kit (Invitrogen) Sequencing was run in the The NextSeq Series desktop sequencing system of the Illumina platform. The number of Pass Filter reads per sample was at the range of (13-14*10^6 reads). Read counts and quality for each sample were evaluated with the BaseSpace Sequencing QC report tool.