RNA-seq analysis of mouse lung

RNA was isolated from C57BL/6 and Stat2^−/− mouse lungs using the Agilent RNA miniprep kit. RNA integrity was determined using an Agilent 2100 bio analyzer. mRNA was purified by using Sera-Mag Oligo(dT) Beads, fragmented with magnesium-catalyzed hydrolysis, and reverse transcribed into cDNA using random primers (Superscript II; Invitrogen). Then, cDNA underwent end repair with T4 DNA polymerase and Klenow DNA polymerase, followed by the addition of “A” bases to the 3′ end and ligation to adaptor oligonucleotides. Products from the ligation were run on a 2% agarose gel. A gel slice consisting of the 200 bp region (±25 bp) was excised and used as a template for PCR amplification. The final PCR product was purified, denatured with 2 N NaOH, and diluted to 10–12 pM prior to cluster amplification on a single-read flow cell v4, as outlined in the Single-Read Cluster Generation Kit v4 (Illumina). The flow cell was sequenced on an Illumina Genome Analyzer II. The data were analyzed as previously described (24 (link)). Full sequencing data has been uploaded to the National Center for Biotechnology Information Gene Expression Omnibus, GSE119029.