Dual index sequencing primers

RNA-seq of primary AT2, AMs, and whole lung tissues was performed by the next-generation sequencing (48 (link)) core in the Division of Pulmonary at Northwestern University or the Laboratory of Xiangdong Fu at University of California, San Diego, as previously described (56 (link), 57 (link)). Briefly, total RNA was extracted and purified using NucleoSpin RNA kit. mRNA was then enriched from total RNA (50 ng) using NEBNext Poly(A) mRNA Magnetic Isolation Module, and complementary DNA (cDNA) libraries were generated using NEBNext Ultra RNA Library Prep Kit for Illumina. The quantity and quality of the cDNA libraries were assessed using an Agilent 4200 Tapestation (Agilent Technologies). cDNA (∼1 ng) was fragmented using high temperature (94° for 15 min) and amplified with a dual-index (i7 and i5; Illumina; 10 cycles), and individual libraries were purified with magnetic beads. Indexed sequence libraries were pooled for multiplexing (∼40 samples per lane), and paired-end sequencing (75 base pairs) were performed on NextSeq 500 using dual-index sequencing primers (Illumina). Reads were aligned using TopHat2 to the mm10 reference genome, differential expression was assessed using edgeR, and enrichment analysis was performed using curated databases including GO or KEGG PATHWAY mapping.