title: GSM2354269: Serum_1. NEBNext; Mus musculus; ncRNA-Seq
library_construction_protocol: Healthy male and female 129/SVj mice, with ages varying between 2 to 9 months, were sedated using tribromoethanol at a dose of 250 mg/kg injected intraperitoneally. 500 μl to 1 ml of blood per mouse was collected in Safe-Lock micro test tubes (Eppendorf) by cardiac puncture using a 0.45 x 12 mm needle, after which mice were sacrificed by cervical dislocation. Clotted blood samples from healthy SVj mice were centrifuged at 1900 g for 10 min at 4 °C using a swinging bucket rotor. The supernatant, containing the serum, was collected and centrifuged again at 16000 g for 10 min at 4°C in a fixed angle rotor to pellet cellular nucleic acids attached to cell debris. 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. Serum samples from individual SVj mice were used as input for RNA isolation using the miRNeasy serum/plasma kit (Qiagen) according to the manufacturer's instructions. Total RNA was eluted in 12 μl of RNase-free water. Small RNA sequencing libraries were prepared using either the TruSeq small RNA library preparation kit v2 (Illumina), the TailorMix miRNA sample preparation kit v2 (Seqmatic) or the NEBNext Multiplex Small RNA library prep kit (New England Biolabs) according to manufacturer's instructions. After PCR amplification, the 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).