Illumina HiSeq 4000 paired end sequencing; RNA-seq analysis of adipose tissue-derived mesenchymal stem cells (AD-MSCs) selected for the surface marker CD271 (CD271+), compared to unsorted stromal vascular fraction (US), and the CD271- AD-MSCs population (CD271-).
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).