P53/ROSA26-Zeb2tg/tg primary mouse T-ALL cells were cross-linked in 1% formaldehyde for 10 min at room temperature and stopped with glycine (final concentration 0.125 M). Cells were lysed in the presence of protease inhibitors and DNA was sonicated. For immunoprecipitation, 100 μg DNA was used together with 3 μl of anti-ZEB2 monoclonal Ab49 (link). Complexes were precipitated with protein A and G Sepharose beads (GE Healthcare). Formaldehyde cross-links were reversed by overnight incubation at 65 °C and DNA was purified using QIAquick PCR Purification Kit (Qiagen). Primers used are given in Supplementary Table 6, including localization of the respective amplicon on the mouse Il7r promotor (Supplementary Fig. 10c). Figure was generated using USCS genome bioinformatics software (http://genome.uscs.edu/).
Goossens S., Radaelli E., Blanchet O., Durinck K., Van der Meulen J., Peirs S., Taghon T., Tremblay C.S., Costa M., Ghahremani M.F., De Medts J., Bartunkova S., Haigh K., Schwab C., Farla N., Pieters T., Matthijssens F., Van Roy N., Best J.A., Deswarte K., Bogaert P., Carmichael C., Rickard A., Suryani S., Bracken L.S., Alserihi R., Canté-Barrett K., Haenebalcke L., Clappier E., Rondou P., Slowicka K., Huylebroeck D., Goldrath A.W., Janzen V., McCormack M.P., Lock R.B., Curtis D.J., Harrison C., Berx G., Speleman F., Meijerink J.P., Soulier J., Van Vlierberghe P, & Haigh J.J. (2015). ZEB2 drives immature T-cell lymphoblastic leukaemia development via enhanced tumour-initiating potential and IL-7 receptor signalling. Nature Communications, 6, 5794.
Other organizations :
VIB-UGent Center for Inflammation Research, Ghent University, University of Milan, Inserm, Hôpital Saint-Louis, Ghent University Hospital, Monash University, Alfred Health, Newcastle University, University of California, San Diego, UNSW Sydney, Walter and Eliza Hall Institute of Medical Research, University of Melbourne, Erasmus MC - Sophia Children’s Hospital, KU Leuven, University of Bonn, Centre Hospitalier Universitaire d'Angers
Cross-linking in 1% formaldehyde for 10 min at room temperature
Immunoprecipitation with anti-ZEB2 monoclonal Ab49
dependent variables
DNA pulled down by anti-ZEB2 antibody
control variables
Glycine (final concentration 0.125 M) was used to stop the formaldehyde cross-linking reaction
Protease inhibitors were used during cell lysis
Protein A and G Sepharose beads were used to precipitate the protein-DNA complexes
Overnight incubation at 65 °C was used to reverse the formaldehyde cross-links
QIAquick PCR Purification Kit was used to purify the DNA
controls
Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned
Annotations
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