GSM5114027: 19-case; Homo sapiens; miRNA-Seq
The total RNA was isolated using the miRNeasy FFPE Kit (QIAGEN®) according to the manufacturer's instructions. Briefly, the paraffin was removed with Xylene and the samples were incubated at 56°C for 15 min, then at 80°C for 15 min in an optimized lysis buffer, which contains proteinase K for RNA release from FFPE sections. Then, the sample was treated with DNase I to eliminate genomic DNA. The lysate was then mixed with guanidine salt-based Buffer to provide appropriate RNA binding conditions   and then ethanol was added, and the samples were transferred to a RNeasy MinElute spin column, where the total RNA binds to the membrane, and contaminants were washed. Finally, the sample containing the total RNA, including miRNA, was eluted in 20 μL of RNase-free water and stored at -80°C. Library preparation was performed from 1 µg of total RNA using the QIAseq miRNA Library Kit from Qiagen. Briefly, a pre-adenylated adapter was ligated to the 3' end of the miRNAs while an RNA adapter was ligated to the 5' of all mature miRNAs. cDNA synthesis was carried out with a reverse transcription primer that binds at the 3' adapter and incorporates a universal molecular index (UMI). The cDNA was cleaned up  using magnetic beads and the library was amplified by using a universal forward primer and indexing reverse primers. The libraries were  cleaned up with magnetic beads and analyzed on an Agilent 2100 Bioanalyzer using a High Sensitivity DNA 1000 chip, according to the manufacturer's instructions. The miRNA Library concentration was determined using the Qubit™ dsDNA HS Assay kit (Invitrogen™), on the Qubit 3.0 Fluorometer (Thermo). The libraries were adjusted to 4nM and pooled (2 10-sample pools) and sequenced at 1 x 75 bp on the MiSeq platform (Illumina) following the instructions of the MiSeq System Denature and Dilute Libraries Guide insert.