Fancd2 antibody

After preclearing with magnetic beads for 1 h, the chromatin from an equivalent of 1 × 10⁷ HCT116 cells was used for immunoprecipitation with a FANCD2 antibody (Novus) or immunoglobulin G as a control. After an overnight incubation at 4 °C, the beads were washed and eluted in buffer E (25 mM Tris–HCl [pH 7.5], 5 mM EDTA, and 0.5% SDS), and crosslinking was reversed at 65 °C with proteinase K for 6 h. The DNA was then purified using a QIAquick PCR purification kit (QIAGEN) and eluted in 100 μl of distilled water. The PCR primer pairs are listed in Supplementary Table S1.

Western immunoblot analysis was performed as described previously^{9 (link)}. Briefly, cells were digested with the cell lysis buffer (Cell Signaling Technology Inc., Danvers, MA, Catalog #: 9803). Protein concentrations were evaluated using the Bradford reagent (Bio-Rad, Hercules, CA, USA). One hundred micrograms of total protein was loaded onto NuPAGE 4–12% Bis–Tris Gel (Invitrogen, Carlsbad, CA, USA). Protein on the gels was electro-transferred onto nitrocellulose membranes and blocked with blocking buffer (5% of non-fat milk, 500 mM of NaCl, 20 mM Tris, and 0.1% Tween 20). The membranes were incubated with primary anti-bodies at 4 °C overnight. After washing with TBS-T (blocking buffer without milk) five times, 10 min each, the membranes were incubated with anti-mouse IgG (Amersham Pharmacia Biotech, Piscataway, NJ, USA, Catalog #: NA931) or anti-rabbit IgG horse radish peroxidase linked to whole secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ, USA, Catalog #: NA934) at room temperature for 1 h. A chemiluminescent detection system (ECLwestern blotting detection reagents, GE) was used to detect the secondary antibody. Finally, the membranes were exposed to x-ray films. Antibodies used were rabbit polyclonal FANCD2 antibody (NovusBiologicals, Littleton, CO, USA, Catalog #: NB100-182) and anti-Actin monoclonal (Sigma, St. Louis, MI, USA, Catalog #: A2228).

ChIP-seq experiments were performed using Active Motif ChIP sequencing services. First, 1 × 10⁷ HCT116 cells that had been treated with or without 0.3 µM APH were fixed in 1% formaldehyde for 15 min. After cell lysis, 30 µg of chromatin was used for immunoprecipitation using a FANCD2 antibody (Novus). Immunoprecipitated and input DNA were sequenced by Illumina sequencing, which generated 75-nt sequence reads. More than 30 × 10⁶ reads per condition were obtained, and a spike-in adjusted normalization method was applied. The peaks were called using the SICER algorithm and aligned to the human genome build hg19. Integrative genomics viewer (IGV) was used to visualize peaks from the genome.