GSM7429593: Omental adipose tissue fibroblasts, mRNA-derived cDNA; Homo sapiens; RNA-Seq
Samples were thawed in a 37ºC water bath and strained over 100µm gauze with PBS to wash out remaining CellSTOR™. Thereafter, adipose explants were extensively mechanically minced and placed into 6 mL pre-warmed (37ºC) adipose tissue media (referred to as adipose buffer hereon in) at pH 7.4 containing 5.5mM D-glucose (Sigma-Aldrich), 140mM sodium chloride, 4.7mM potassium chloride, 1.25mM magnesium sulfoxide heptahydrate, 2.5mM calcium chloride dihydrate, 10mM HEPES, 2.5mM sodium phosphate monobasic dihydrate, and 2 % bovine serum albumin (BSA) fraction V, spiked with type-1 collagenase (250U/mL) (Worthington Biochemical). Samples were agitated at 180–200 rpm on a orbital shaker () at 37ºC for 45–60 minutes. Digestions were terminated with 2 volumes of PBS containing 10 % FBS with 2mM ethylenediaminetetraacetic acid (EDTA). Following digestion, samples were passed through a 400 μm gauze and centrifuged at 100 x g for 1 minute at RT, whereupon the adipocyte fraction was aspirated and discarded. The remaining post-digestion solution was centrifuged at 300 x g for 7 minutes at RT, and was thereafter incubated with 9 volumes erythrocyte lysis buffer for 10 minutes at RT. Post-lysis, samples were topped up with adipose buffer, centrifuged once more at 300 x g for 7 minutes at RT and the supernatant discarded before topping up with adipose buffer for filtering through 100 μm gauze, and centrifuged once more at 300 x g for 7 minutes at RT before being resuspended in PBS containing 2mM EDTA and 2 % FBS (referred to hereon in as FACS buffer). Following stromal cell isolation cell sorting was undertaken on a BD™FACSAria III cell sorter (BD Biosciences) to separate CD45+/- events. Library generation was performed according to the manufacturer's protocol (Chromium Single Cell 3ʹ kit (10x Genomics).