GSM3692197: P1_Low; Homo sapiens; RNA-Seq
Human adipose tissue samples were digested in a collagenase 2 buffer to release the stromal vascular fraction (SVF) from mature adipocytes. The SVF cells were stained with fluorescent antibodies specific to CD45, CD31, CD29 and CD34. The human adipocyte precursor cells (APCs) within the SVF were identified in the lineage negative fraction (CD31- CD31-) as CD29+ CD34neg, CD29+ CD34low, CD29+ CD34high cell types and were isolated in a FACS instrument. The isolated cells were frozen immediately, and total RNA was extracted from the frozen cells by using RNeasy Mini kit (Qiagen, Germany). RNA quality of the samples was further assessed by measuring the RNA integrity number (RIN) using the Agilent 2100 Bioanalyzer (Agilent Biosystems) and the RNA concentration was assessed in a Qubit Bioanalyzer (ThermoFisher Scientific). The RNA samples were first amplified using the Ovation RNA-Seq system V2 kit (NuGEN, protocol M01206v5, 2013) in a thermocycler and then subjected to cDNA synthesis. Single Primer Isothermal strand-displacement Amplification (SPIA) technology was adopted to synthesize cDNA. A total of 15 cDNA libraries were constructed from the RNA samples by this process. The RNA-seq libraries were then constructed from the cDNA samples by using the Ovation Ultralow System V2 (NuGEN) as per the product instructions. An equimolar pool (12 pM) of the all the ligated sample libraries, ~274 bp in size on an average, were pooled and hybridized onto two lanes of the HiSeq 1500 high-output flowcell per sample.
GEO Accession
GSM3692197