GSM2552691: T8.D.2670558; Homo sapiens; miRNA-Seq
Peripheral blood samples were collected in PAXgene blood RNA tubes (PreAnalytix). PAXgene tubes were frozen using a sequential freezing process: tubes were stored at room temperature for 3hrs, transferred to 4°C overnight, followed by 6-8 hrs at -20°C, then final storage at -80°C. Total RNA (including the miRNA fraction) was isolated from whole-blood using the PAXgene Blood miRNA Kit (Qiagen, Canada), according to manufacturer’s instructions. RNA and miRNA yield and quality were determined using the Nanodrop 1000 (Thermo Scientific, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA). All libraries were prepared using the Illumina TruSeq Small RNA protocol following the manufacturer's instructions with 12 cycles of PCR amplification after ligation of the 3' and 5' adapters, as described elsewhere5. Libraries were purified using biotinylated magnetic AMPure beads that allow for selection of specified cDNA products bound to streptavidin. A total of 50µl of amplified cDNA were mixed and purified twice with AMPure XP beads at a 1.8:1 ratio (beads:sample). This ratio allows for optimal selection of all products higher than 100nt. Library qualities were assessed using an Agilent 2100 Bioanalyzer High Sensitivity DNA chip, as well as qRT-PCR with the KAPA library quantification kit (Kapa Biosystems, USA). Samples were sequenced at the McGill University and Genome Quebec Innovation Centre (Montreal, Canada) using the HiSeq2500 Illumina sequencer with 50nt single-end reads.
GEO Accession
GSM2552691