GSM2299009: L-33-30622637; Homo sapiens; RNA-Seq
ASCs and fibroblasts were detached from culture plates by trypsin digestion at confluence and cell numbers were counted using Countess Automated Cell Counter (Invitrogen, Carlsbad, CA).  Approximately 500,000 cells from each sample were collected and frozen at -80oC until RNA isolation.  Total RNA was extracted from the cells using TRIzol (Thermo Fisher Scientific, Grand Island, NY) and quality was examined by electrophoresis. Poly (A)+RNA were isolated from the total RNA samples using the NEBNext Poly (A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA).  RNA-seq libraries were generated for each sample from 1 ug of mRNA using the NEBNext Ultra Directional RNA Library Prep Kit with NEBNext® Multiplex Oligos for Ilumina according to the manufacture’s protocol (New England Biolabs, Ipswich, MA).  Size distribution of the libraries was determined using the Agilent Bioanalyzer High Sensitivity Chip on Agilent Bioanalyzer 2100 (Santa Clara, CA).  The concentration of the libraries were quantified by quantitative PCR using the the KAPA SYBR® FAST ABI Prism qPCR Kit and Applied Biosystems® 7500 Real-Time PCR System (Thermo Fisher Scientific, Grand Island, NY).  The libraries were pooled and diluted to 1.8 pM with sequencing reagents from the Illumina NextSeq 500/550 High Output kit and sequenced on the NextSeq 500 desktop sequencer (Illumina Inc., San Diego, CA)