Chromatin Immunoprecipitation of Gli1 and Acetyl-H3

ChIP was performed using the MAGnify Chromatin Immunoprecipitation System (Invitrogen). Protocol was performed as described in^{44 (link)}. For each ChIP reaction 300,000 cells were used, cell lysates were added to their respective antibody/beads for 2 h. Eluted DNA was PCR amplified with primers encompassing the Gli- responsive elements of human ABC promoter. The following antibodies were used: IgG rabbit (Invitrogen), rabbit polyclonal anti-Gli1 H300 (sc-20687, Santa Cruz Biotechnology Inc.), rabbit polyclonal anti-acetyl-histone 3 (06599, Millipore). Eluted DNA has been analysed with Q-PCR. Primers were designed with Primer-Blast designing tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and Primers tool (Genomatix Genome Analyzer, GGA, v3.30126, https://www.genomatix.de/) and are reported in Supplementary Table 2.

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Po A., Citarella A., Catanzaro G., Besharat Z.M., Trocchianesi S., Gianno F., Sabato C., Moretti M., De Smaele E., Vacca A., Fiori M.E, & Ferretti E. (2020). Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells. Scientific Reports, 10, 13988.

Publication 2020

MAGnify Chromatin Immunoprecipitation System IgG rabbit anti-Gli1 H300 anti-acetyl-histone 3

Antibodies Antibody Cell Chip Chromatin immunoprecipitation Genome Histone Human Primers Rabbit

Corresponding Organization :

Other organizations : Sapienza University of Rome, Istituto Superiore di Sanità, Istituto Pasteur

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Variable analysis

independent variables

Antibody used for ChIP (IgG rabbit, rabbit polyclonal anti-Gli1 H300, rabbit polyclonal anti-acetyl-histone 3)

dependent variables

Enrichment of Gli-responsive elements in the human ABC promoter, measured by Q-PCR

control variables

Cell number (300,000 cells per ChIP reaction)
Incubation time of cell lysates with antibody/beads (2 hours)
Primer design (designed with Primer-Blast and Primers tool)

controls

Negative control: IgG rabbit

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