RNA-seq of mus musculus: primary lung fibroblasts
Total RNA was extracted from the lung fibroblasts isolated from Il11ra-/- and Il11ra+/+ mice using RNeasy columns (Qiagen). The protocols for primary mouse lung fibroblast isolation and culture were previously described (1). RNA was quantified using a Qubit RNA Broad range Assay kit (Life Technologies) and its quality was assessed on the basis of their RNA integrity number using the Bioanalyzer RNA 6000 Nano Assay (Agilent Technologies). Libraries prepared using the TruSeq Stranded mRNA Kit (Illumina) were pooled and sequenced on a NextSeq 500 sequencer (Illumina) using NextSeq 500 High Output v2 kit and paired-end 75base pair sequencing chemistry. Sequenced libraries were demultiplexed using bcl2fastq v2.19.0.316 and adapters trimmed using trimmomatic 6 v0.36. Reads were aligned to GRCm38 (mm10) using STAR v2.5.2b and only unique alignments were retained for counting using feature counts. Read coverage for Galt, Il11ra1, Ccl27a were visualized with IGV using strand specific alignment (BAM) files.