organism: Homo sapiens
description: Protocols: 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: ENA-FIRST-PUBLIC
attributes.value: 2020-02-27T04:03:39Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-02-17T16:58:15Z
attributes.tag: External Id
attributes.value: SAMEA6565206
attributes.tag: INSDC center name
attributes.value: Cardiff University, School of Medicine
attributes.tag: INSDC first public
attributes.value: 2020-02-27T04:03:39Z
attributes.tag: INSDC last update
attributes.value: 2020-02-17T16:58:15Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8803:AG02
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: rectal adenocarcinoma
attributes.tag: fraction
attributes.value: EpCAM+CD3-Live+
attributes.tag: individual
attributes.value: patient 1
attributes.tag: organism part
attributes.value: rectum
attributes.tag: sample name
attributes.value: E-MTAB-8803:AG02
attributes.tag: sampling site
attributes.value: normal tissue far from neoplasm
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: male