Qubit fluorometric quantification kit

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).

Lumbar spinal cord samples were obtained from SOD1^WT, SOD1^G93A EfnA5^+/+ and SOD1^G93A EfnA5^+/− mice at 130 days of age after cervical dislocation, and were lysed by addition of lysis buffer containing 6 M guanidine hydrochloride, 10 mM Tris (2-carboxyethyl)phosphine hydrochloride (TCEP), 40 mM 2-chloroacetamide and 100 mM triethylammonium bicarbonate (TEAB). Samples were then sonicated, heated at 95 °C for 10 min and centrifuged at 20000 g for 30 min at 4 °C, and the supernatant was collected for further analysis. Protein concentration was determined using the Qubit Fluorometric Quantification Kit (Thermo Scientific). Protein digestion was performed with a filter-aided sample preparation (FASP) protocol and analysed by LC-MS/MS as previously described [4 (link)]. Proteins were identified using MaxQuant 1.5.2.8 and the Mus musculus reference proteome from UniProt (downloaded 12th March 2018). A false discovery rate (FDR) of 1% was used for peptide and protein identification (total protein IDs after exclusion of contaminants: 5525) and protein quantification was performed with the MaxLFQ algorithm [8 (link)]. Quantitative analysis of the data was performed with Perseus 1.5.2.6 [40 (link)]. Proteins with valid values in at least three samples per group were used for the quantitative comparison (4065 proteins).