Rabbit igg clone da1e

Around 3-4 x 10⁶ cells were crosslinked with 1% PFA at room temperature for 10 minutes, quenched with 125 mM Glycine, and rinsed twice with PBS. Cells were lysed and sonicated at 4°C using a Bioruptor Sonicator (Diagenode, UCD-200TM) into 100-600 bp fragments. For STAT1 ChIP, Dynabeads Protein A (Invitrogen, 10002D) was pre-coated with 10 μg anti-STAT1 (clone D1K9Y, Cell Signaling Technology, cat#14994S) or Rabbit IgG (clone DA1E, Cell Signaling Technology, cat#3900S) antibody. For H3K27ac ChIP, Dynabeads Protein A (Invitrogen, 10002D) was pre-coated with 5 μg anti-H3K27ac (clone D5E4, Cell Signaling Technology, cat#8173S) or Rabbit IgG (clone DA1E, Cell Signaling Technology, cat#3900S) antibody. The samples were incubated with antibody pre-coated beads at 4°C overnight. ChIP samples were then washed, eluted, and reverse crosslinked by overnight incubation at 65 °C. ChIP-DNA was purified by Phenol:Chloroform:Isoamyl Alcohol (Sigma-Aldrich, 77677-100ML) and used for qPCR analysis. ChIP enrichment was calculated relative to input samples. Primers used for ChIP-qPCR are listed in Supplementary Table 3.

Sheared chromatin for ChIP analysis was generated using the ChIP-IT Express Kit (Active Motif, Carlsbad, CA, USA). Fifteen micrograms of chromatin were pre-cleared with Protein G magnetic beads (Cell Signaling) and isotype control antibodies, mouse IgG (clone G3A1, Cell Signaling) or rabbit IgG (clone DA1E, Cell Signaling), for 1 h at 4˚C. Immunoprecipitation with 5 μg of specified antibodies against NME1 (SC-NM301, Santa Cruz Biotechnologies), Histone 3 lysine 27 acetylation (H3K27ac, clone D5E4, Cell Signaling), Histone 3 lysine 4 trimethylation (H3K4, Cell Signaling), and RNA Polymerase II (Active Motif) was performed overnight at 4˚C on an end-to-end rotator. Eluted DNA was purified using the QiaQuick PCR Purification Kit (Qiagen) prior to qPCR analysis, using specified primer sequences found in Table I. Quantification was achieved by normalization to input DNA, and expressed as % input.