GSM3476352: 0074.C2; Homo sapiens; miRNA-Seq
Blood samples were collected in EDTA tubes, then, plasma samples were obtained by a single centrifugation (1000g, 15min. at room temperature). For RNA extraction from 200ul of plasma, miRCURY RNA insolation kit-Biofluids (#300112) was used following manufacturer indications with modifications that included using glycoingen as carrier and a 1:50 dilution of spike in RNA. Barcoded sequencing libraries were prepared using the TrueSeq SmallRNA library kit (Illumina) with slight modifications from the default recommendations (Part # 15004197 Rev. F, February 2014). Modifications included using all RNA extracted from 200 ul of plasma, in the subnanogram range, and increasing the number of cycles in the final PCR reaction (from 12 to 15). Libraries were quality assessed with Bioanalyzer DNA 1000 assays (Agilent). Bioanalyzer concentration estimates were used to generate an equimolar library mix. The library pool was automatically size selected (135-160bp) using 3% agarose dye free cassettes with 3% DF marker F on a Pippin Prep system with software v.6.0 (Sage Science). QIAquick (Qiagen) column purifications were performed prior and after to size selection. The library pool was quantifiedby PCR with theKAPA Library Quantification Kit following manufacturer's instructions (Roche). The library pool was run on 4 lanes of 1x50 bp sequencing was performed on HiSeq2500 platform (Illumina) using adapted protocols for TruSeq SR cluster kit v3 (Part#15023335) following cBot™ (Part#15006165) and HiSeq2500 System (Part#15035786) user guidelines. Raw data were analysed by Real Time Analysis software v. 1.17.21.3 and Sequence Analysis Viewer software v. 1.8.20 (Illumina.).
GEO Accession
GSM3476352