Diamag protein a coated magnetic beads

Cells were lysed in RIPA lysis buffer (MilliporeSigma) supplemented with protease inhibitor (MilliporeSigma) and 10 mM N-ethylmaleimide. Lysates (1.25 mg of protein per sample) were diluted in 0.5% NP-40 buffer, precleared with DiaMag protein A-coated magnetic beads (Diagenode), and incubated with 2 μg of RPA194 primary antibody overnight at 4 °C. Samples were incubated the next day with DiaMag protein A-coated magnetic beads, and the bead–antibody–protein complexes were washed extensively with 0.5% NP-40 buffer. The bead–antibody–protein complexes were resuspended in 35 μl of Laemmli sample buffer (Bio-Rad) and DTT, and the samples were boiled and run on a NuPAGE 3 to 8% Tris-Acetate gel. Along with the input, the precipitated protein was transferred to a PVDF (0.45 μm) membrane and probed first for FK2 ubiquitin (MilliporeSigma; catalog no.: 04-263) and then for RPA194. As a negative control, one sample was incubated overnight with 2 μg of normal mouse IgG (MilliporeSigma; catalog no.: 12-371) rather than the RPA194 primary antibody.

Primary HDFs overexpressing ANKRD1 were previously cross-linked for protein-protein interactions with Ethylene glycol bis(succinimidyl succinate) (EGS) at a final concentration of 1.5 mM 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 (400 g). 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 a 100-300 bp range using Diagenode Bioraptor. Samples were precleared using the beads included in the kit and incubated overnight at 4 °C with 5 µg of 10 µl of commercially available V5 tag antibody from GENETEX targeting the V5 tag in ANKRD1 expressing fibroblasts. Non-immune controls with non-immune IgG were included. Antibody–chromatin complexes were pulled down using protein A-beads from the kit (DIAmag protein A-coated magnetic beads, DIAGENODE). Elution was performed according to the manufacturer’s instructions. Chromatin was quantified using the Qubit Fluorometric Quantification Kit (ThermoFisher Scientific).

ChIP was carried out as described before with some modifications (63 (link), 64 (link)). Briefly, cells were cross-linked by adding 1% (w/v) formaldehyde and incubated for 20 min at RT with gentle shaking. Cell fixation was interrupted by adding 110 mM glycine. Cells were scraped off and resuspended in lysis buffer following the iDeal ChIP-qPCR kit protocol (Diagenode, Liège, Belgium). To obtain genomic DNA fragments between 500 and 100 bp, cell lysates were sonicated for four rounds of ten cycles (30 s ON/30 s OFF) using the Bioruptor Pico (Diagenode) at high power setting. For immunoprecipitations, the following antibodies were used: 1 μg of rabbit polyclonal anti-IgG (C15410206, Diagenode) as negative control IP; 1 μg of mouse monoclonal anti-FOXJ1 (14-9965-82, Thermo Fisher Scientific) and 3.8 μg of mouse monoclonal anti-FLAG (F1804, Sigma Aldrich). Chromatin–antibody complexes were immunoprecipitated by DiaMag Protein A-coated magnetic beads (Diagenode). DNA isolation and de-cross-linking was carried out as described by the iDeal ChIP-qPCR kit protocol (Diagenode). Coprecipitated DNA was quantified by real-time qPCR using the primers listed in Table S2.