title: GSM4656204: EGR1_ChIP_Kasumi-1_shRE_plusDox; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were crosslinked in 1% formaldehyde for 10 minutes before being quenched by 0.4M Glycine. For EGR-1 ChIP, cells were also double-crosslinked with Di(N-succinimidyl) glutarate (DSG) for 45 minutes. Cells were lysed for 10 minutes in cell lysis buffer (5mM PIPES pH 8.0, 85mM KCl, 0.5% NP-40, 1:200 Protease Inhibitory Cocktail [PIC] before undergoing nuclear lysis for 10 minutes in nuclear lysis buffer (50mM Tris pH 8.0, 10mM EDTA, 1% SDS, 1:100 PIC). Nuclei were then sonicated in a Bioruptor Pico (Diagenode) until chromatin was sheared to a 200-600bp fragment size and then diluted in blocking buffer (16.7mM Tris pH 8.0, 167mM NaCl, 1.2mM EDTA, 1.1% Triton X-100, 0.01% SDS and 1:100 PIC). 15ul of Dynabeads G were pre-blocked for 2 hours at 4oC with blocking buffer consisting of 0.5% BSA, 30µg glycogen and ChIP grade antibody at a suitable concentration, before being incubated overnight 4oC with the diluted chromatin. See Key Resources table for details of antibodies used. Then the beads were washed in sonication wash buffer (50mM Tris pH 8.0, 10mM EDTA, 1% SDS), twice in Buffer A (20mM Tris, pH 8.0, 0.1% SDS, 1% Triton-X 100, 2mM EDTA, 150mM NaCl), twice in Buffer B (20mM Tris, pH 8.0, 0.1% SDS, 1% Triton-X 100, 2mM EDTA, 150mM NaCl), with Lithium buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA pH 8.0, 0.5 % NP-40 and 0.5 % sodium deoxycholate). Beads were then washed once in 1xTE. Chromatin was then eluted in 100mM NaHCO3 and 1% SDS and reverse crosslinked for 4-16 hours at 65oC with 50μg Proteinase K. DNA was then cleaned up with 1.8x SPRI beads. qPCR amplification of immunoprecipitated DNA was compared to input at several loci of putative protein binding sites as well as non-binding sites to assess the success of ChIP. See Supplementary file 5 for qPCR primer sequences. Libraries for high throughput sequencing were prepared using the Kapa HyperPrep kit (Kapa biosystems, USA), as per manufacturer's protocol. 16 cycles of PCR was performed and 200-400 bp fragments were size selected by running the samples in an agarose gel. Libraries were validated by qPCR, with an analysis of the ChIP signal of a positive control region (e.g. PU.1 14 3H enhancer) over a negative control region (e.g. IVL). Finally, libraries were quantified by Kapa library quantification kit (Kapa biosystems, USA) and run in a pool of four indexed libraries in one lane of a HiSeq 2500 (Illumina, USA) or 12 indexed libraries in one lane of a NextSeq 500 (Illumina, USA) using 50 cycle single-end reads.