Instrument: NextSeq 500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: The tissue was collected fresh and sterile from the operating room and transported on ice to the pathologist in the absence of formalin fixative. Following pathological examination, tissue in excess of diagnostic requirements was collected and subsequently de-identified at the tissue bank. The tissue was then transported on ice to the laboratory. Samples that were collected in the evening were stored in media at 4C overnight. The duration from surgery to the commencement of tissue dissociation did not exceed 24 hrs, the majority of the samples were processed on the same day as surgery. Fragments of normal ER+ breast tissue were of insufficient cell number to utilize FACS after they were dissociated as described above, so epithelial cell enrichment was performed by utilizing magnetic EasySEP EpCAM beads (StemCell Technologies). Once the cells were washed in cold PBS, the appropriate amount of selection media and magnetic particles was added and incubated on ice for 15 minutes. Next, the tube was placed on the magnet at room temperature for five minutes and eluted with chilled media and this process was repeated for a total of four times to enrich for the ER+/EpCAM+ epithelial tumor cells. Immuno-labelling of normal cells was performed at 4C using PBS as a buffer and wash solution. Incubating the cell pellet with 0.1 mg/ml DNase, 1 mg/ml rat immunoglobulin and a 1/10 dilution of anti-CD16/CD32 receptor antibody for 10 min. was sufficient to block non-specific antibody binding. The cell suspension was then made up to a final concentration of 106 cells /40 l with dilution buffer. For primary human breast tissue, antibodies directed against human lineage markers CD31 (endothelial cells), CD45 (leukocytes) and CD235a (red blood cells) were used to prepare a Lin- cell population, and these PE-linked antibodies were negatively selected against. Primary antibodies were added at optimized dilutions and incubated for 25 min at 4C. The suspension was then washed, filtered using 40uM tube cap filters (Falcon) and centrifuged. Cells were resuspended in 0.5 g/ml propidium iodide (Sigma) before cell analysis and sorting to allow for only the selection of viable cells. Cell analysis and sorting was performed on either a FACS DIVA or FACS Aria (BD Biosciences) using a sheath pressure of 30 psi and nozzle size of 70 um. Cells were sorted into polystyrene FACS tubes (Westnet #352063) and collected into a mixture of 50% primary MEC media and 50% CDT. Reanalysis of the sorted populations routinely revealed a purity of more than 95%.