GSM4407252: US-1-3; Mus musculus; RNA-Seq
Mice were sacrificed at days 1, 7, 28 or 90 days after exposure by overdose via intraperitoneal injection of 2 mL pentobarbitone. At all considered time points, the lungs were carefully dissected from the large airways and small portions of each lobe were extracted for RNA extraction. Tissue samples were placed in RNAlater (Invitrogen, Thermo Fisher Scientific, UK), and placed immediately on ice. Samples in RNAlater solution were incubated overnight at 4 degree Celsius prior to storage at -20 degree Celsius, until further processing. Tissue samples in RNAlater solution (Invitrogen, Thermo Fisher Scientific, UK) were homogenised using a TissueLyser LT (QIAGEN, UK). Homogenised samples were then loaded onto spin cartridges containing silica membranes, for extraction of total RNA performed using a PureLink RNA Mini kit (Invitrogen, Thermo Fisher Scientific, UK) according to the manufacturer's instructions. Total RNA concentration and purity were calculated by measuring the optical density at 230, 260 and 280 nm, using a Biophotometer Plus spectrophotometer (Eppendorf AG, Germany). RNA quantity and RNA integrity number (RIN) were assessed by Agilent 2200 bioanalyzer. Sample RNA with RIN values below 8 was discarded. The isolated RNA samples were stored at -80°C until RNA sequencing was carried out. Illumina TruSeq Stranded mRNA Library Prep Kit (Illumina, Inc., San Diego, CA, USA) was used to prepare mRNA sequencing libraries. Briefly, 1000 ng of total RNA was used for mRNA isolation using poly dT-coated beads.