GSM2344863: NCr Mouse 1047. TimePoint 1.tRNA dep; Mus musculus; miRNA-Seq
100 µl blood was collected by puncture of the jugular vein using a 4 mm lancet and collected in a BD Vacutainer collection tube with a gel separator (BD). All blood samples clotted at room temperature for 45 min. Clotted blood samples were centrifuged at 2000 g for 15 minutes at 4°C in a fixed angle rotor. The supernatant was collected and stored at -80 °C. For all serum samples, the degree of hemolysis was determined by measuring levels of free hemoglobin by spectral analysis. Absorbance peaks at 414, 541 and 576 nm are indicative of free hemoglobin. RNA was isolated from 50 μl of serum that was collected from Ncr nude mice and eluted in 12 μl of RNase-free water using the miRNeasy serum/plasma kit (Qiagen) according to the manufacturer's instructions. For RNA samples that are depleted for tRNA-halves, biotinylated DNA probes complementary to the 5' tRNA-halves were allowed to hybridize to the target RNA sequences. Magnetic streptavidin beads were used to immobilize the DNA-RNA complexes. Using a magnetic field, the beads and bound DNA-RNA complexes were separated, after which the supernatant, containing tRNA-depleted RNA, was collected and purified using ethanol precipitation and suspended in 7.5 µl RNase-free water. Small RNA sequencing libraries were prepared using the TruSeq Small RNA Library Preparation kit v2 (Illumina) according to manufacturer's instructions using 6 µl of RNA as input. After PCR amplification (15 cycles), quality of libraries was assessed using a high sensitivity DNA kit on a Bioanalyzer (Agilent) according to manufacturer's instructions. Size selection was performed using 3% agarose dye-free marker H cassettes on a Pippin Prep (Sage Science) following manufacturer's instructions with a specified collection size range of 125-153 bp. Libraries were further purified and concentrated by ethanol precipitation, resuspended in 10 μl of 10 mM Tris-HCl (pH=8.5) and quantified using qPCR. Based on the qPCR results, equimolar library pools were prepared, quality was assessed as described above and the library was further diluted to 4 nM using 10 mM Tris-HCl (pH=8.5).