Antibodies and reagents
Plasmids and siRNAs
Cell culture and transfection
SILAC labeling
Nuclear extraction
Protein extraction and trypsin digestion
HPLC fractionation
Kcr peptide enrichment
Database searching and protein quantification
Bioinformatics analysis
Immunoprecipitation and Western blotting
Purification of recombinant proteins
GST pull-down assay
Detection of ssDNA
Immunofluorescence staining and confocal microscopy
DNA-protein binding assay
Electrophoretic mobility shift assay
Colony formation assay
Flow cytometry
Statistical analysis
The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a homemade reversed-phase analytical column (15 cm length, 75 μm ID). The gradient was composed of an increase from 6 to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23 to 35% in 8 min, and climbing to 80% in 3 min and then holding at 80% for the last 3 min, all at a constant flow rate of 400 nl/min on an EASY-nLC 1000 ultra performance liquid chromatography (UPLC) system. The peptides were subjected to nanospray ionization (NSI) source followed by MS/MS in Q Exactive Plus (Thermo Fisher Scientific) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z (mass/charge ratio) scan range was 350 to 1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using normalized collision energy (NCE) setting as 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure alternated between one MS scan followed by 20 MS/MS scans with 15.0-s dynamic exclusion. Automatic gain control was set at 5 × 104. Fixed first mass was set to 100 m/z.