GSM6625438: ANFX8298; Mus musculus; RNA-Seq
Whole blood samples were collected into Tempus™ Blood RNA tubes (Thermo Fisher Scientific, Waltham, MA, USA) or PAXgene blood tubes (Qiagen, Hilden, Germany) containing RNA stabilizing reagent. Total RNA was isolated using QuickGene blood cell kit (RB-S) and Fujifilm QuickGene-810 automated system (Fujifilm Life Sciences, Tokyo, Japan) or PAXgene Blood miRNA Kit and fully automated QIAcube system (Qiagen), according to manufacturer's isolation protocol. The purity of isolated RNA was assessed using the NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific) and integrity was determined on Agilent 2100 Bioan+alyzer using RNA 6000 Nano LabChip kit (Agilent Technologies, Santa Clara, CA, USA). All samples used for library preparation had RNA integrity values greater than 7.0. Extracted RNA samples were stored at -80ºC before further processing. We performed library preparation using the TrueSeq stranded total RNA sample preparation kit (Illumina, San Diego, CA, USA), according to the manufacturer's protocol. After Ribo-Zero rRNA depletion, the remaining RNA was purified, fragmented, and primed for first strand cDNA synthesis with reverse transcriptase (SuperScript II) and random primers, followed by second strand cDNA synthesis performed in the presence of dUTP. Blunt ended double strand DNA was 3' adenylated and multiple indexing adapters (T-tailed) were ligated to the ends of the ds cDNA. Fragments with adapter molecules on both ends were selectively enriched with 15 cycles of PCR reaction. 44 libraries were normalized to the final concentration of 10 nM and then pooled in equimolar concentrations. Sequencing was performed on the Illumina Hiseq 2000 sequencing system in 2×100 sequencing cycles using pair-end sequencing mode.