title: GSM2419819: RELA ChIP-seq No Treatment Batch 6; Homo sapiens; ChIP-Seq
library_construction_protocol: After treatment, cells were crosslinked with 0.75% formaldehyde for 8 minutes and cross-linking was quenched with 180mM Glycine for 10 minutes at room temperature. Cells were then lysed with cell lysis buffer (0.25% Triton X-100, 10mM EDTA, 0.1M NaCl in 10mM Tris-HCl pH8) for 20 minutes at 4°C. Nuclei were collected by centrifugation at 4,000rpm for 10 minutes at 4°C and lysed in nuclei lysis buffer (1% NP-40, 0.5% NaDOC, 0.1% SDS in PBS) for 20 minutes at 4°C. Chromatin shearing was performed using a sonicator (Bioruptor, Diagenode) on high setting for 45 cycles of 30 seconds ON/45 seconds OFF, leading to a fragment range of 100-250bp. ChIP-seq libraries were built following the library-on-beads protocol from Wallerman et al. (Wallerman et al. 2015). Chromatin was incubated with anti-RELA antibody (NFKB p65 (c20): sc-372) bound to protein G magnetic beads (Dyanabeads protein G, Thermo Fisher scientific) prepared by mixing 4ug of antibody with 40uL of beads in PBS+0.5%BSA for 4 to 6 hours at room temperature. Immunoprecipitation was performed by rotation at 4°C overnight. After immunoprecipitation, samples were washed twice with nuclei lysis buffer, twice with nuclei lysis buffer complemented with 350mM NaCl, twice with LiCl/NP-40 buffer (0.25M LiCl, 1mM EDTA, 0.5% NP-40, 0.5% NaDOC in 10mM Tris-HCl pH8) and once with PBS. Libraries on beads were then built using NEBnext DNA library prep kit (New England Biolabs) according to the manufacturer’s instructions. Due to the nature of the samples, minor adjustments were done: two washes with PBS were performed in between each step of the preparation and elution of the DNA from the beads was done after adaptor ligation. ChIP samples were eluted in Elution buffer (10mM EDTA, 1% SDS in 50mM Tris-HCl pH7.5) for 4 hours at 65°C and purified with Agencourt AMPure XP beads (Beckman Coulter). PCR reaction was performed using Phusion High-Fidelity PCR Master Mix (Thermo scientific) and custom primers for multiplexing. The following program was applied: 3 minutes at 98°C; 17x(30 seconds at 98°C, 30 seconds at 65°C, 30 seconds at 72°C); 5 minutes at 72°C. Samples were loaded on a DNA1000 bioanalyzer chip (Agilent) to check the size of the libraries as well as the concentration. As a control, 2-5% of input DNA was reverse cross-linked at 65°C for minimum 6 hours and libraries were prepared using the same library preparation kit.  Samples were sequenced on an Illumina HiSeq Rapid 2x76bp.
attributes.tag: GEO Accession
attributes.value: GSM2419819