Mouse anti myc tag clone 4a6

To identify the genomic sites that are bound by HOXC9, we performed two independent ChIP-seq assays with BE(2)-C/Tet-Off/myc-HOXC9 cells cultured in absence of doxycycline for 6 days according to the published procedure [11 ]. Briefly, 4 × 10⁷ cells were fixed with 1% formaldehyde for 10 min and quenched with 0.125 M glycine for 5 min. After cell lysis, cross-linked chromatin DNA was sheared to approximately 250 bp by sonication (Model 150E ultrasonic dismembrator, Fisher Scientific), and immunoprecipitated with 10 μg of mouse anti-myc tag (clone 4A6, Millipore) and 80 μl Dynabeads Protein G (Invitrogen). The immunoprecipitated HOXC9-DNA complexes were washed extensively and eluted with SDS buffer, followed by incubation overnight at 65°C to reverse cross-linking. The samples were then treated sequentially with RNase A and proteinase K to degrade associated RNA and proteins, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Input genomic DNA was purified from an aliquot of chromatin after sonication. DNA concentration was determined using a PicoGreen dsDNA quantitation assay kit (Invitrogen).