Illumina HiSeq 1500 sequencing; ChIPseq of DDX3X, DDX54, ER and H3K4me3 in human MCF7 cells
ChIPseq of DDX3X, DDX54, ER and H3K4me3 in human MCF7 cells
MCF7 cells were grown in standard conditions in 15 cm diameter dishes to confluence and cross-linked with 1% (v/v) formaldehyde for 15 minutes at room temperature and cross-linking was stopped by the addition of 0.125M glycine. Cells were then lysed in 1% [v/v] SDS, 10mM EDTA, 50mM Tris-HCL pH8.0, 1mM and protease inhibitors. Cells were sonicated in QSONICA sonicator (1h cycle 5 secs ON/5 secs OFF) to achieve a mean DNA fragment size of 500bp. Antibodies used are: DDX54 Rabbit polyclonal Abcam- ab76947; DDX3 Mouse monoclonal Abcam-ab196032; Mouse Control IgG2a Isotype Control Abcam-ab18413; Rabbit IgG, polyclonal Isotype Control Abcam-ab27478; ERa (Estrogen Receptor alpha) Rabbit monoclonal Abcam ab32063; H3 (tri methyl K4) Rabbit polyclonal Abcam- ab8580; Rabbit IgG polyclonal Isotype Control Abcam-ab27478 Chromatin was pre-cleared by incubation with protein A/G dynabeads magnetic beads (Thermo Scientific) in  modified RIPA buffer (1% [v/v] Triton-X- 100, 0.1% deoxycholate, 0.1% SDS, 90mM NaCl, 10mM Tris-HCL pH8.0, 1mM sodium orthovanadate and EDTA-free protease inhibitors) in rotation at 4°C for 2 hours. Immunoprecipitation (IP) was performed for a minimum of 12 hours at 4°C in modified RIPA buffer with anti-DDX3, anti-DDX54, anti-ER, anti- H3K4me3 and anti-IgG Rb isotype control antibodies. All IPs were performed in triplicate. Protein A (for rabbit antibodies) and G (for mouse antibodies) previously equilibrated in modified RIPA buffer, were used to bind the antibody and associated chromatin for a minimum of 2 hours in rotation at 4°C. The beads were then washed twice with wash buffer (0.1% [v/v] SDS, 1% [v/v] Triton-X-100, 2mM EDTA, 20mM Tris pH8.0, 150mM NaCl) and once with final wash buffer (0.1% [v/v] SDS, 1% [v/v] Triton-X-100, 2mM EDTA, 20mM Tris pH8.0, 500mM NaCl). DNA was then eluted with 200μl of elution buffer (1% [v/v] SDS, 100mM NaHCO3) for 15 minutes at room temperature. The input and immunoprecipitated samples were then mixed with 2μl of RNase (DNase free-Roche) for a minimum of 5 hours at 65°C to reverse the crosslinks. DNA purification was performed using the ChIP DNA ZYMO purification kit.  Purified DNA was used for preparation of ChIP-seq libraries with NEXTflex ChIP-Seq Library Prep Kit for Illumina Sequencing from Bioo Scientific, following manufacturer instructions. Purified DNA was used for preparation of ChIP seq libraries with NEXTflex ChIP-Seq Library Prep Kit for Illumina Sequencing from Bioo Scientific, following manufacturer instructions.
Experimental Factor: immunoprecipitate
anti-H3K4me3 (#ab8580 Abcam)