title: Illumina HiSeq 2000 paired end sequencing; RNA seq of rectal tumour tissue and paired healthy tissue, with EpCAM purification
design: RNA seq of rectal tumour tissue and paired healthy tissue, with EpCAM purification
library_name: AG09_p
library_construction_protocol: Tumour and paired background (unaffected) colon specimens were obtained from three patients undergoing anterior resection for primary rectal cancer. Within 30-minutes of resection from a patient, samples were minced with blades in a Petri dish and forced through 70mm cell strainers to collect a single cell suspension. In no instances were collagenase or DNase treatments used. Dissociated cell preparations of tumor, near and far healthy  colonic tissue were initially stained with the amine-reactive viability dye Live/Dead fixable Aqua (ThermoFisher) followed by surface marker staining with CD3-APC (BioLegend) and EpCAM-PE (Miltenyi Biotec) antibodies. Samples were resuspended in FACS buffer (PBS, 2% BSA) prior to sorting into Live/Dead-EpCAM+CD3- populations on a FACS Aria III (BD). Tumor tissue also stained with CD3 and EpCAM antibodies was additionally passed through the cell sorter without gating, and used as an unsorted control for RNA-sequencing. All samples were sorted directly into RLT buffer (Qiagen) with b-mercaptoethanol (Sigma Aldrich) and frozen at -80˚C. Frozen samples were thawed and RNA isolated using an RNA easy micro isolation kit (Qiagen). Library preparation and RNA sequencing was carried out by VGTI-FL (Florida, USA). Purified RNA was used to make libraries using an Illumina TruSeq kit.
attributes.tag: Experimental Factor: sampling site
attributes.value: normal tissue near to neoplasm
attributes.tag: Experimental Factor: fraction
attributes.value: EpCAM+CD3-Live+