title: MEFwt_anti-Sp2-Exo
organism: Mus musculus
description: Protocols: 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
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-10-17T17:03:27Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-08-09T14:52:33Z
attributes.tag: External Id
attributes.value: SAMEA4824132
attributes.tag: INSDC center name
attributes.value: INSTITUTE FOR MOLECULAR BIOLOGY AND TUMOR RESEARCH
attributes.tag: INSDC first public
attributes.value: 2018-10-17T17:03:27Z
attributes.tag: INSDC last update
attributes.value: 2018-08-09T14:52:33Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7125:MEFwt_anti-Sp2-Exo
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: mouse embryonic fibroblast cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: embryo
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: embryo
attributes.tag: sample name
attributes.value: E-MTAB-7125:MEFwt_anti-Sp2-Exo
attributes.tag: scientific_name
attributes.value: Mus musculus