title: GSM3711775: AT-12-VAT; Homo sapiens; RNA-Seq
library_construction_protocol: Adipose tissue was digested with collagenase according to a modification of the Robdell method (Tchernof et al., 2006). Briefly, adipose tissue samples were digested with collagenase type 1 in Krebs-Ringer-Henseleit (KRH) buffer for 45 minutes at 37°C. Cell suspensions containing mature adipocytes and the stromal-vascular fraction (SVF) were then filtered with a nylon mesh and washed 3 times with KRH buffer. The nature of the buoyancy adipocytes allows them to float to the surface. Mature adipocytes were discarded and the remaining solution containing the SVF was centrifuged 1500 rpm for 5 minutes. The pellet was washed with pre-adipocyte growth medium (PGM) (DMEM-F12 supplemented with 10% calf serum, 1% penicillin-streptomycin, 17µM pantothenic acid, 33µM biotin, 100µM ascorbic acid and 2,5 µm/ml amphoB) followed by a second centrifugation. Cells were cryopreserved using freezing medium (PGM supplemented with 40% FBS and 10% DMSO). The medium was added to the pellet and was frozen with a temperature gradient (-1 °C/ minutes) and store at -80°C until analysis. The FACS sorted SVF samples were incubated with primary antibody (CD34) and sorted using FACSAria II Cells from the SVF were thawed and serially diluted in DMEM/F12 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in 15ml conical tube to a volume of 14ml. They were centrifuged at 300 rcf for 10 minutes and the supernatant was removed and discarded. The pelleted cells were suspended in an adequate amount of DMEM/F12 supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin and transferred to a 1.5ml microfuge tube for counting. 10 µl of the suspension was used for a cell count and viability assay on a Countess II Automated Cell Counter (Invitrogen) to determine the volume of suspension to be used in the 10x Chromium Single Cell Library protocol. The desired number of viable cells to be loaded onto the Chromium Single Cell A Chip (10x Genomics) was 12,000 cells. After cell capture, the remaining cells were centrifuged at 300 rcf for 10 minutes and cryopreserved using freezing medium (Recovery Cell Culture Freezing Medium, Thermo Fisher Scientific). The cells were suspended in freezing medium and frozen with a temperature gradient of -1⁰C per minute. Cell capture, cDNA amplification, and library preparation were performed using the Chromium Single Cell 3' Library & Gel Bead Kit v2 (10x Genomics), according to the manufacturer's protocol. The final libraries were analyzed using a TapeStation to determine library size and a Qubit Broad Range dsDNA assay to determine library concentration and sequenced using Illumina HiSeq. Chromium Single Cell 3' Library
attributes.tag: GEO Accession
attributes.value: GSM3711775