Chromatin Immunoprecipitation for Nkx2-1 Targets

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)).

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Herriges M.J., Tischfield D.J., Cui Z., Morley M.P., Han Y., Babu A., Li S., Lu M., Cendan I., Garcia B.A., Anderson S.A, & Morrisey E.E. (2017). The NANCI–Nkx2.1 gene duplex buffers Nkx2.1 expression to maintain lung development and homeostasis. Genes & Development, 31(9), 889-903.

Publication 2017

EpCAM antibody anti-APC microbeads LS columns

Adult Antibody Cells Chip Epcam Epithelial cells Genome Human Igg antibody Lung Microbeads Mouse Nkx2 1 Primers Rabbit

Corresponding Organization :

Other organizations : University of Pennsylvania, Children's Hospital of Philadelphia, Philadelphia University, California Institute for Regenerative Medicine, Cardiovascular Institute of the South

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Variable analysis

independent variables

EpCAM antibody (eBioscience, 17-5791-82)
Anti-APC microbeads (Miltenyi Biotec, 130-090-855)
LS columns (Miltenyi Biotec, 130-042-401)
Sonication using Bioruptor for 8 min (30 sec on, 30 sec off)
Nkx2.1 antibody (Millipore, 07-601)

dependent variables

Nkx2.1-bound DNA
QPCR results on isolated samples

control variables

Adult lung epithelial cells
Rabbit IgG antibody

controls

Positive control: Samples isolated using Nkx2.1 antibody
Negative control: Samples isolated using rabbit IgG antibody

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