title: NEG_patient2
organism: Homo sapiens
description: Protocols: Abdominal adipose tissue was harvested from donor patients undergoing DIEP flap breast reconstruction at Wythenshawe Hospital, Manchester University NHS Foundation Trust, UK, after informed consent was granted. Ethics approval was given under 13/SC/0499. Adipose tissue was mechanically dissociated and digested with collagenase I for 1 hour at 37ºC. The resulting digested tissue was filtered through a 100µm nylon mesh and centrifuged at 160g for 10. The pellet (the stromal vascular fraction, SVF) was treated with Red Blood Cell Lysis Buffer to remove contaminating red blood cells prior to Magnetic-Activated Cell Sorting (MACS). MACS was carried out using CD271 microbeads, producing three populations (unsorted, CD271+, CD271-). The SVF obtained from the homogenised fat samples was sorted via Magnetic-Activated Cell Sorting (MACS) protocol. Some cells were kept as unsorted populations (US) before resuspending the remainder in MACS buffer (0.5% Bovine Serum Albumin and 2mM Ethylenediaminetetraacetic Acid in Phosphate Buffer Saline solution), CD271 microbead solution (Miltenyi Biotec, Surrey, UK, 130-099-023) and FcR blocking reagent for 15 minutes at 4°C. The cells were then washed with MACS buffer, centrifuged at 300g for 10 minutes, and resuspended in MACS buffer prior to addition to the magnetic column attached to a MACS magnet (Miltenyi Biotec, Surrey, UK, 130-090-976). The column was repeatedly washed with MACS buffer and the cell suspension that passed through the column was collected as the negative population (CD271- AD-MSCs). Then the column was removed from the magnet and MACS buffer was added to the column, and the plunger depressed; this flow-through was collected as the positive population (CD271+ AD-MSCs). RNA was extracted from AD-MSCs using a Qiagen RNeasy plus mini kit (Qiagen, Manchester, UK), according to manufacturer protocol. cDNA was transcribed from RNA using Qiagen RT2 reverse transcription kit as per datasheet (Qiagen, Manchester, UK). Library preparation of samples was performed using the Illumina TruSeq Stranded mRNA Sample Preparation Kits (Illumina).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2021-01-22T04:07:20Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-01-27T16:09:25Z
attributes.tag: External Id
attributes.value: SAMEA6504167
attributes.tag: INSDC center name
attributes.value: UNIVERSITY OF MANCHESTER
attributes.tag: INSDC first public
attributes.value: 2021-01-22T04:07:20Z
attributes.tag: INSDC last update
attributes.value: 2020-01-27T16:09:25Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8740:NEG_patient2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: adipose tissue derived mesenchymal stem cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: individual
attributes.value: 2
attributes.tag: organism part
attributes.value: adipose tissue
attributes.tag: phenotype
attributes.value: CD271-
attributes.tag: sample name
attributes.value: E-MTAB-8740:NEG_patient2
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: female