Lungs from Avertin anesthetized mice were harvested after perfusion with cold PBS through the right ventricle. Extra-pulmonary tissues were then removed and lungs were minced using forceps and crosslinked with 1:4 dilution of 10% buffered formalin (ThermoFisher Scientific, 23-245-685) in PBS on a rocker at room temperature for 10 minutes. Samples were then quenched by adding 1M glycine (pH5) to a final concentration of 125mM and incubated on a rocker for 10 minutes at room temperature. Samples were washed two times with 2mL of PBS and resuspended with 1mL of isolation of nuclei tagged in specific cell types (INTACT) (20mM Hepes pH7.4, 25mM KCl, 0.5mM MgCl2, 0.25M sucrose, 1mM DTT, 0.4% NP-40, 0.5 mM Spermine, 0.5mM Spermidine) with protease inhibitor cocktail (cOmplete ULTRA Tablets, Mini, EDTA-free, EASY pack, Roche) (Mo et al., 2015, Deal and Henikoff, 2010). Resuspended samples were then homogenized using an electric homogenizer with three pulses lasting five seconds each. The resulting solution was then filtered through a 70µm cell strainer and centrifuged in a 2mL tube at 384 rcf for 5 minutes. The pellet was resuspended in a PBS with protease inhibitor cocktail (cOmplete ULTRA Tablets, Mini, EDTA-free, EASY pack, Roche, 05 892 791 001). Nuclei were counted and 1 million nuclei aliquots were added to six 1.7mL tubes (Olympus plastics, 24-282). These subdivided samples were then centrifuged down at 384 rcf for 5 minutes. The pellet was resuspended in 100µL ChIP nuclei lysis buffer (50mM Tris-HCl pH 8.1, 10mM EDTA, 1% SDS with 1x protease inhibitor cocktail). After incubation on ice for fifteen minutes, samples were then sonicated using a Bioruptor Twin (Diagenode, UCD-400-TO) in a 4°C cold room on the high setting for 37 cycles of 30 seconds on 30 seconds off to achieve DNA fragment size of 200-500bp. The samples were then centrifuged at 12,052 rcf for 15 minutes at 4°C. The supernatant from each tube was pooled to a total volume of 600µL. From this pooled chromatin, 20µL of the sample was taken as an input control, diluted with Tris-EDTA (TE) buffer (1mM EDTA, 10mM Tris-HCl pH 8.1), and stored at -80°C. The remaining 580µL of chromatin was diluted to 1mL with ChIP dilution buffer (16.7mM Tris-HCl pH 8.1, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS with 1x protease inhibitor cocktail) and precleared with 40µL Protein G Dynabeads (ThermoFisher Scientific, 10004D) in a 2mL tube for 1 hr on a rotator at 4°C. Precleared chromatin divided into four tubes (250µL/tube) and diluted to 1mL with ChIP dilution buffer such that roughly 1 million nuclei were incubated with 1 µg of each Nkx2-1 antibody overnight at 4°C on a rotator. Concurrently, 30µL protein G Dynabeads for each antibody were blocked overnight at 4°C on a rotator in 500µL ChIP dilution buffer with the addition of 200µL 25mg/mL Bovine Serum Albumin (Jackson ImmunoResearch, 001-000-161), and 4µL 10mg/mL Salmon DNA Sperm (Invitrogen, 15632-011). The next morning, the blocked beads were washed with ChIP dilution buffer twice and transferred to new 2mL tubes. The antibody-chromatin solution was added and incubated for 3 hr at 4°C while rotating. The dynabeads were then isolated using a magnetic adaptor and washed with 1 mL of each of the following buffers for five minutes each on a rotator: low salt buffer (150mM NaCl, 2mM EDTA, 1% Triton X-100, 20mM Tris-HCl pH 8.1, 0.1% SDS), high salt buffer (500mM NaCl, 2mM EDTA, 1% Triton X-100, 20mM Tris-HCl pH 8.1, 0.1% SDS), lithium chloride buffer (250mM LiCl, 1mM EDTA, 1% NP-40, 10mM Tris-HCl pH 8.1, 0.1% sodium deoxycholate) twice and TE buffer twice. The resuspended sample after the second TE wash was transferred to fresh 2mL tubes and a magnetic adaptor was used to collect the dynabeads and 300µL fresh TE was added. The samples and the frozen input chromatin were then incubated at 37°C with 1.5µL of 10mg/mL RNase A (Qiagen, 1007885) for 1 hr, followed by a 4 hr incubation at 55°C after adding 3.5µL of 20mg/mL Proteinase K (ThermoFisher Scientific, EO0491) and 15µL of 10% SDS. The incubation continued overnight at 65°C to reverse the crosslinking. DNA extraction was then performed by adding 1 volume of phenol–chloroform-isoamyl solution (Sigma, P2069-400ML) followed by centrifugation at 12,052 rcf for 15 minutes. DNA in the upper phase were precipitated by adding 2 volumes of 100% ethanol, 1/10 volume of 3M NaCl and 3µL of 20 µg/µL glycogen (Invitrogen, 10814-010). The solution was then vortexed briefly and centrifuged for 30 minutes at 12,052 rcf in a 4°C cold room. The pellet was washed with 70% ethanol without resuspension. The sample was then centrifuged for 5 minutes at 9,200 rcf at room temperature. The pellet was air dried for 10 minutes and dissolved in 10µL nuclease free ddH2O. ChIP DNA quantity was measured using the Qubit dsDNA HS Assay Kit (Invitrogen, Q32851). 2-5ng of sample DNA was used to generate sequencing libraries using the NEB Next Ultra II DNA Library Prep Kit for Illumina (NEB, E7645). Step 3.1 in the NEB protocol was skipped to improve sample retention per manufacturer's recommendation. Samples were PCR amplified for 12 cycles and barcoded using indexing primers (New England BioLabs, E7335s or E7500S) followed by a final (.65 x -1 x bead volume) size selection and purification step using a SPRIselect reagent (Beckman Coulter, B23318). Samples were then verified for library size by gel electrophoresis and concentration measured using the Qubit HS dsDNA assay. Samples were then sequenced on the Illumina NextSeq500.