GSM9009478: RNA-Seq, PBMCs, HFD + L-NAME + NO2-OA 6; Mus musculus; RNA-Seq
PBMCs were thawed according to 10x Genomics demonstrated protocol #CG00039. RNA was purified from cells using the miRNeasy kit (Qiagen, #217004) according to manufacturer's instructions with additional DNase (Qiagen, #79254) digestion. Quality control and quantification of the isolated RNA were performed using Fragment Analyzer 5200 (Agilent) using the High Sensitivity RNA quantification kit and Fragment Analyzer Controller Software (Agilent v3.1.0.12). Random primed library preparation was started with 150 ng of total RNA using the Watchmaker RNA library prep kit with Polaris depletion (Watchmaker Genomics) according to the instructions of the manufacturer. The barcoded libraries were purified and quantified using Qubit Fluorometric Quantification (ThermoFisher Scientific). Size distribution of the library DNA was analyzed using the Fragment Analyzer 5200 (Agilent). Sequencing of 2x150 bp was performed with an Illumina NovaSeq 6000 sequencer (Illumina, San Diego, CA, USA).