title: GSM6736618: Input; Homo sapiens; ChIP-Seq
library_construction_protocol: Primary human dermal fibroblasts overexpressing ANKRD1 were previously cross-linked for protein-protein interactions with ethylene glycol bis succinimidyl succinate (EGS) at a final concentration of 1.5mM for 30 minutes. Formaldehyde was added to a final concentration of 1% for 10 min at RT. The reaction was quenched using the addition of glycine (final concentration 125 mM). Cells were washed with ice-cold PBS and collected by centrifugation. Next, cells  were lysed using the iDeal ChIP-seq kit (DIAGENODE) for Transcription Factors according to the manufacturer's instructions DNA in the cross-linked chromatin was fragmented by sonication to 100-500 bp range using Diagenode Bioraptor. Samples were precleared using the beads included in the kit and incubated overnight at 4°C with 5 ug of commercially available V5 tag antibody from GENETEX targeting the V5 tag in ANKRD1 over-expressing fibroblasts. Incubations of parallel samples performed Non-immune controls with non-immune IgG. Antibody–chromatin complexes were pulled down using protein A- beads from the kit (DIAmag protein A-coated magnetic beads, DIAGENODE). Elution proceeded according to the manufacturer's instructions. Chromatin was quantified using the Qubit Fluorometric Quantification Kit (Thermo Fisher Scientific). ChIP-Seq library preparation and data analysis: as recommended by the manufacturer, a total of 10 ng of ChIPed DNA was used for library preparation using NEBNext® ChIP-Seq Library Prep Reagent Set for Illumina. Sequencing was carried out at the Lausanne Genomic Technologies Facility (GTF) using the HiSeq 4000.