RNA sequencing

For bulk RNA-sequencing experiments, whole foregut tissue was dissected from E11.5 Nkx2-1^-/- or WT embryos, and lungs were removed at the time of dissection. For RNA-sequencing of wild type (WT) trachea and esophagus, trachea and esophagus were manually separated at the time of dissection. Foreguts of individual embryos were dissociated, stained, and sorted as described above. Each biological replicate consisted of RNA pooled from two Nkx2-1^-/- or WT embryos, with a total of 3 biological replicates from different litters. RNA was purified using the RNeasy Micro kit (Qiagen, cat# 74004) and quantification was performed using the RNA 6000 Pico kit (Agilent, cat# 5067–1513) on an Agilent 2100 Bioanalyzer. RNA-sequencing libraries were prepared from 4 ng of input RNA using the SMARTer Stranded Total RNAseq kit V2 (Takara, cat# 634411) with 13 amplification cycles. Library size and quality was checked using an Agilent 2100 Bioanalyzer with the High Sensitivity DNA kit (Agilent, cat# 5067–4626), and library concentration was determined with the QuBit dsDNA HS Assay kit (Invitrogen, cat# {"type":"entrez-protein","attrs":{"text":"Q32854","term_id":"75280861","term_text":"Q32854"}}Q32854). Libraries were normalized to 7 nM, pooled, and sequenced across two lanes of an Illumina HiSeq 4000 to generate 50 base pair single-end reads. Data processing and downstream analysis was performed as described below.