Spri ampure xp beads

ChIP-seq was performed as previously described^{52 (link)}. Briefly, chromatin samples prepared using the appropriate number of fixed cells (3 × 10⁵ for histone modifications and 5 × 10⁶ for transcription factors) were sonicated and subsequently immunoprecipitated with each antibody recognizing 5 μg of CTCF (Cell Signaling Technology, 2899), 5 μg of SMC1 (Bethyl lab, A300-055A), 5 μg of RelA (Cell Signaling Technology, 8242), 5 μg of STAT5 (Cell Signaling Technology, 9351), 1 μg of H3K27ac (Abcam, ab4729), 1 μg of H3K4me1 (Abcam, ab8895), 1 μg of H3K4me3 (Abcam, ab8580), or 1 μg of H3K27me3 (Abcam, ab6002). Antibody-chromatin complexes were captured with protein A and G Dynabeads (Invitrogen, 100.02D/100.04D), washed with Low Salt Wash Buffer, High Salt Wash Buffer, and LiCl Wash Buffer. Chromatin-antibody immobilized on magnetic beads were then subjected to tagmentation. Eluted DNA was purified using SPRI Ampure XP beads (Beckman Coulter, A63881) and amplified for 8–12 cycles using Nextera PCR primers. Libraries were purified using dual (0.5x–2.0x) SPRI Ampure XP beads and paired-end sequenced (100 bp) on the Illumina HiSeq2500 platform. Two biological replicates were performed for each condition.

A total of 1×10⁶ cells/mL were fixed with 1% formaldehyde at room temperature for 10 minutes, washed with PBS, and stored at −80 °C. Cells were sheared with the Chromatin EasyShear Kit–Low SDS (Diagenode, Belgium) and sonicated with a Bioruptor Pico sonication device (catalog No. B01080010, Diagenode, Belgium) according to the manufacturer’s recommendations. The MAGnify Chromatin Immunoprecipitation System (Thermo Scientific) was used to select chromatin fragments using anti–histone H3 (trimethyl K4; ab8580) or anti-histone H3 (acetyl K27; ab4729). Sequencing libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit according to manufacturer’s instructions. In brief, purified ChIP or input DNA was end repaired and A-tailed followed by adaptor ligation. The adaptor-ligated DNA was size selected with SPRI Ampure XP beads (Agencourt) and enriched with Illumina-compatible sequencing primers. The final libraries were purified with SPRI Ampure XP beads to remove adaptor dimers and sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 3000/4000 platform and the 50-bp single-read configuration.