Bulk RNA-seq Analysis of Lung Fibrosis

For bulk RNA-seq analysis of lung fibrosis, bleomycin (3 U/kg; Hospira) or water was instilled intratracheally to anesthetized male 129S1 mice, age 10–12 weeks. Cells gated on SiglecF⁺CD11c⁺ and sorted on MHCII^hi or MHCII^lo (Supplementary Fig. 6a) were collected at 2 weeks and 4 weeks after injury, or at baseline for the MHCII^lo compartment. RNA was collected (RNeasy Micro, Qiagen) from these samples, and total RNA quality was assessed by spectrophotometer (NanoDrop, Thermo Fisher Scientific Inc., Waltham, MA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Intact mRNA was isolated using the Dynabead mRNA Purification Kit for total RNA, according to manufacturer’s protocol (Thermo Fisher Scientific, Waltham, MA). Amplified cDNA was prepared using the NuGen Ovation RNA-Seq system V2 kit, according to the manufacturer’s protocol (NuGen Technologies, Inc., San Carlos, CA), and sequencing libraries were generated using the Nextera XT library preparation kit with multiplexing primers according to manufacturer’s protocol (Illumina, San Diego, CA). Library fragment size distributions were assessed using the Bioanalyzer 2100 and the DNA high-sensitivity chip (Agilent Technologies, Santa Clara, CA). Library sequence quality was assessed by sequencing single-end 50 base pair reads using the Illumina MiSeq platform, and libraries were pooled for high-throughput sequencing on the Illumina HiSeq 4000 by using equal numbers of uniquely mapped protein coding reads. RNA sequencing was performed on HiSeq 4000 machines (Illumina, San Diego, CA), followed by de-multiplexing of raw sequencing results, trimming of adapter sequences, and alignment to the reference genome using STAR software^{49 (link)}. DESeq2 was used to normalize by size factor (reads per sample) as well as by library complexity, and we then applied Wald test to determine significance of differential expression.