Epcam antibody

E18.5 and P4 lungs were harvested and processed into single-cell suspensions using a dispase (Collaborative Biosciences)/collagenase (Life Technologies)/DNase solution. Epithelial cells were enriched using an EpCAM antibody (eBioscience, 17-5791-82) and Dynabeads (Invitrogen, sheep anti-rat IgG) following the manufacturer's instructions. Adult lungs (4 wk, 10 wk, and 6 mo) were harvested, and total RNA was isolated using Trizol (Invitrogen) per the manufacturer's protocol. cDNA was synthesized from total RNA by using the SuperScript strand synthesis system (Invitrogen). qPCR was performed using the SYBR Green system (Applied Biosystems) with primers listed in Supplemental Table 5. GAPDH expression values were used to control for RNA quality and quantity.

ChIP:PCR was performed as described previously (Snyder et al. 2013 (link)). Adult lung epithelial cells were isolated from a single-cell suspension using an EpCAM antibody (eBioscience, 17-5791-82) in conjunction with anti-APC microbeads (Miltenyi Biotec, 130-090-855) and LS columns (Miltenyi Biotec, 130-042-401). These cells were sonicated using a Bioruptor for 8 min (30 sec on, 30 sec off), and Nkx2.1-bound DNA was purified using an Nkx2.1 antibody (Millipore, 07-601) and the MAGnify ChIP kit following the manufacturer's instructions. qPCR was performed on the isolated samples using primers listed in Supplemental Table 4 and normalized to samples isolated using a rabbit IgG antibody. The mouse genome (mm9; human hg19) was scanned for the occurrence of Nkx2-1 motifs using the FIMO program from the MEME suite (Grant et al. 2011 (link)).

For RNA-Seq and ChIP-Seq analyses, IECs were isolated as follows: mice were sacrificed and colonic specimens were dissected, opened longitudinally and cleared from feces by washing extensively in cold PBS. Colons were cut in small pieces of 1 mm and incubated in 30 mM EDTA solution in PBS at 37 °C for 10 min. 10 min later, EDTA solution was replaced with ice-cold PBS and shacked vigorously for 30 s. This process was repeated once more. Supernatants were collected and combined from both incubations, then centrifuged at 1200 rpm for 5 min at 4 °C. For culture purpose, the colons were cut into pieces and washed by DMEM for three times, and then incubated with digestion buffer containing 250 μg/ml collagenase type I (Sigma, C0130) and 500 μg/ml Dispase II (Roche #04942078001) for 1.5 h in 37 °C. After the incubation, the cell suspension was passed through 100 μm cell strainers (Corning). After washes, the cells were plated in dishes coated with rat tail tendon collagen type I overnight and were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin. The purities of colonic epithelial cell from the EDTA-based and enzymatic digests were 90–95% and 80–85%, respectively, using an EpCAM antibody (Invitrogen #25-5791-80) and FACS analysis.