Sample 18
Homo sapiens
Protocols: 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.
ENA first public
2019-11-14
ENA last update
2017-11-14
External Id
SAMEA104379785
INSDC center alias
Gurdon Institute, University of Cambridge
INSDC center name
Gurdon Institute, University of Cambridge
INSDC first public
2019-11-14T04:02:57Z
INSDC last update
2017-11-14T15:48:13Z
INSDC status
public
Submitter Id
E-MTAB-6211:Sample 18
broker name
ArrayExpress
cell line
MCF-7
cell type
epithelial cell
common name
human
disease
invasive ductal carcinoma
metastatic site
pleural effusion
organism part
mammary gland
sample name
E-MTAB-6211:Sample 18