SD_Lung_6
Total RNA from lung tissue samples was extracted using TRIzol and the RNeasy Mini Kit (Qiagen). Lung tissue was mechanically disrupted in 700 l of TRIzol and 140 l of chloroform was added and the sample thoroughly mixed. Following a 3 min incubation at RT, the sample was then centrifuged for 15 min at 15,000 xg, 4C. The clear top layer was then carefully collected, mixed with an equal volume of 70% ethanol and RNA extracted using the RNeasy Mini Kit, with on-column DNase digestion, following the manufacturers instructions. RNA was eluted in water and RNA concentration and quality were measured using a TapeStation system (Agilent) with the High Sensitivity RNA ScreenTape assay. mRNA purification and cDNA synthesis for the sequencing library were performed according to the Illumina Stranded mRNA Prep protocol (20040534) using the following index kit: IDT for Illumina RNA UD Indexes Set A, Ligation (20040553). Quality and concentration of the final libraries were checked with the KAPA Library Quantification Kit (Roche Diagnostics) in a StepOnePlus thermal cycler (Applied Biosystems) according to manufacturers instructions. All cDNA libraries were sequenced using a paired-end strategy (read length 150 bp) on an Illumina NovaSeq platform.