title: GSM4042945: PMD000018_Cap25; 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 150um nylon mesh and washed 3 times with Hank's Balanced Salt Solution (HBSS) supplemented with 200um adenosine and 2% FBS and centrifuged at 200 rcf for 8 minutes. The nature of the buoyancy adipocytes allows them to float to the surface during the centrifugation step. Mature adipocytes were discarded and the remaining solution containing the SVF was centrifuged 300 rcf for 6 minutes. The pellet was washed with HBSS+200um adenosine+2%FBS followed by centrifugation. The pellet was treated with ACK lysis buffer at RT for 5 minutes, followed by another centrifugation step. The ACK lysis buffer was removed from the pellet and the cells were washed once more with HBSS+200uM adenosine+2%FBS and centrifuged one final time. Cells were counted to determine the total number of cells in suspension and viability. Cells were then cryopreserved using freezing medium (Gibco™ Recovery™ Cell Culture Freezing Medium, 10% DMSO). The medium was added to the pellet and was frozen with a temperature gradient (-1 °C/ minutes) and stored in liquid nitrogen until analysis. 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 two wells of a Chromium Single Cell A Chip (10x Genomics) was 8000 cells for a total of two captures with 4000 cells/capture. 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 and stored in liquid nitrogen. 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 two libraries were analyzed using a TapeStation to determine library size and a Qubit Broad Range dsDNA assay to determine library concentration and sequenced on Illumina NovaSeq6000 instrument.
attributes.tag: GEO Accession
attributes.value: GSM4042945