title: GSM2333689: HUVEC_Scr_IL1beta_FA_Input; Homo sapiens; ChIP-Seq
library_construction_protocol: For Notch1, RBPJ, and NF-κB/RelA ChIP-seq experiments, cells were crosslinked in a two-step crosslinking procedure: 45 min in 2 mM disuccinimidyl glutarate (DSG) followed by 10 min incubation with 1% formaldehyde (FA) before quenching with 125 mM glycine. For H3K27ac ChIP-seq, only FA crosslinking was applied. For each 15 cm dish, cells were lysed in 1.6 mL lysis buffer (0.1% SDS, 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 20 mM Tris pH 8), removed with a cell scraper, and kept at 4°C before sonication (material used for NF-κB/RelA and H3K27ac ChIP-seq was stored at -80°C). DSG/FA and FA samples were sonicated for 40 and 16 cycles, respectively, on a Bioruptor Twin set to 30 sec ON/OFF and high intensity. To remove cell debris, samples were subjected to centrifugation at 10,000g for 1 min and supernatants were collected. ChIP was performed on approximately 1.5e7 cells in a final volume of 6 mL lysis buffer with BSA in 15 mL tubes. Chromatin was incubated with antibodies as indicated in the list above for 3 hours at 4°C with upside-down rotation. Protein A sepharose beads were added and samples were incubated overnight at 4°C with upside-down rotation. Beads were washed in 12 mL by the following wash steps: 2x wash buffer 1 (0.1% SDS, 0.1% NaDOC, 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 20 mM HEPES), 1x wash buffer 2 (0.1% SDS, 0.1% NaDOC, 1% Triton X-100, 0.5 M NaCl, 1 mM EDTA, 20 mM HEPES), 1x wash buffer 3 (0.25 M LiCl, 0.5% NaDOC, 0.5% NP-40, 1 mM EDTA, 20 mM HEPES), and 2x wash buffer 4 (1 mM EDTA, 20 mM HEPES). Samples were treated with 10 µg RNase A in 100 µL for 20 min at 37°C. Elution was carried out in 2x200 µL elution buffer (0.1M NaHCO3, 1% SDS) for 2x15 min at room temperature with upside-down rotation. Samples were then treated with 60 µg Proteinase K for 1 hour at 55°C and subjected to decrosslinking in 300 mM NaCl by overnight incubation at 65°C. DNA was purified by standard PCI/chloroform extraction and ethanol precipitation and resuspended in 60 µL molecular biology-grade water. For input samples, 0.7% sonicated material was processed in parallel to the ChIP-seq samples from the RNase-treatment step. Sequencing libraries were prepared according to the Illumina protocol, and sequenced on an Illumina Hiseq4000 (RBPJ, Notch1) or Illumina Nextseq500 (RelA, H3K27ac) machine at the Genomics Core Facility at Oslo University Hospital, Norway.
attributes.tag: GEO Accession
attributes.value: GSM2333689