RNA-Seq of mus musculus: adult male lung
Total RNA was extracted from samples using a RNeasy Micro Plus Kit (Qiagen). Total RNA integrity was determined using Agilent Bioanalyzer or 4200 TapeStation. Library preparation was performed with 10 ng total RNA with a Bioanalyzer RNA integrity number score greater than 8. Double standard complementary DNA (cDNA) was prepared using the SMARTer Ultra Low RNA kit for Illumina Sequencing (Takara-Clontech) per manufacturers protocol. cDNA was fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, and cycles per burst 50 for 120 s. cDNA was blunt ended, had an A base added to the 3 ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12 to 15 cycles using primers, incorporating unique dual index tags.