RNA-Seq of Mus musculus: adult male lung
Total RNA (300 ng) underwent rRNA depletion with Ribo-Zero Gold (Human/Mouse/Rat) and (Yeast) kit (Illumina, San Diego, CA). RNase III fragmentation and adaptor ligation were performed using PrepX mRNA Library kit (Takara, Mountain View, CA). cDNA synthesis utilized Superscript III reverse transcriptase (Lifetech, Grand Island, NY) and purification with Agencourt AMPure XP beads (Beckman Coulter, Indianapolis, IN). Universal and index-specific primers were used for barcoding, followed by PCR and library enrichment with AMPure XP beads. Library quality and yield were assessed by Bioanalyzer (Agilent, Santa Clara, CA) and qPCR using Kapa Library Quantification kit (Kapa Biosystems, Woburn, MA) on ABI's 9700HT real-time PCR system (Lifetech, Grand Island, NY). Final libraries (12.0 pM) were clustered on a flow cell with Illuminas TruSeq SR Cluster kit v3 and sequenced for 50 cycles using TruSeq SBS kit on Illumina HiSeq system.