Illumina HiSeq 1500 sequencing; ChIP-seq of Pbx1, Prep1 and Sp2 in WT MEFs, Sp2ko MEFs and in Sp2ko MEFs ectopically expressing Flag-tagged full length Sp2; and ChIPexo of Sp2 in WT and  Sp2ko MEFs
ChIP-seq of Pbx1, Prep1 and Sp2 in WT MEFs, Sp2ko MEFs and in Sp2ko MEFs ectopically expressing Flag-tagged full length Sp2; and ChIPexo of Sp2 in WT and  Sp2ko MEFs
cell culture, see growth and nucleic acid extraction protocol. Antibodies used: Anti-Pbx1 (Cell Signaling Technology, Danvers, USA, Cat. No. #4342); Anti-Prep1 (Santa Cruz, Dallas, USA, Cat. No. sc-25282); antiSp2 Ab1 (produced in-house) MEFs were cultured in a 1:1 mixture of Dulbecco's modified eagle medium (DMEM)/high glucose (4.5 g/L) (Gibco Thermo Fisher Scientific, Waltham, USA) and HAM's F-10 (Gibco Thermo Fisher Scientific, Waltham, USA) supplemented with 10% (v/v) fetal calf serum (Sigma-Aldrich, St. Louis, USA) and 1% Penicillin-Streptomycin. Sp2ko MEFs expressing Sp2 mutants were selected and propagated in the presence of 2 µg/mL puromycin. For ChIP-exo analysis we used the ChIP-exo kit from Active Motif (Carlsbad, CA) in accordance with the manufacturer´s instructions. Cross-linked chromatin was generated from 2x107 wild type and Sp2ko MEFs. Three individual ChIP reactions with 500 microgram of sheared chromatin were performed for each cell type. Six microgram of affinity-purified homemade Sp2 antibody #1 were used per ChIP experiment. Enzymatic reactions and DNA purification steps were performed as described in the ChIP-exo kit manual. After PCR amplification, reactions were pooled and purified with AMPure magnetic beads (Beckman Coulter, Brea, CA). The DNA library was size selected by agarose gel electrophoresis (size range 180-350 bp) and extracted DNA fragments were purified with QIAquick columns (Qiagen, Hilden, Germany). Acitve Motif ChIP-exo kit
Experimental Factor: immunoprecipitate
anti-Sp2
Experimental Factor: genotype
wild type genotype