GSM5235420: rs03_Adequate_Sleep; Homo sapiens; RNA-Seq
For mRNA extraction, the harvested endotheial cell pellet was re-suspended with isolation buffer, incubated with biotinylated mouse anti-human monoclonal antibody directed against CD146 (Millipore, Temecula, CA,1:200) at 4°C for 15 min, then incubated at 4°C with Streptavidin FlowComp Dynabeads (Invitrogen, Solo, Norway; 1:100) for 45 min, followed by EC isolation by magnet. mRNA was extracted using RNAqueous Micro Kit (Invitrogen by Thermo Fisher Scientific) and used for constructing sequencing libraries. Total RNA harvested from endothelial cells at the end of adequate sleep and sleep restriction was converted and amplified to cDNA using Ovation Pico WTA System V2. Amplified cDNA was then used to prepare sequencing libraries using the Ultra II DNA Library Preparation Kit (NEBNext). Briefly, mRNA was prepared from the total RNA by generation of first strand cDNA using primers containing oligo dT, which selects for the 3' poly A tails of mRNA. The samples are fragmented and reverse transcribed into cDNA. The ends are repaired by addition of an “A” base and ligated to adapters which enable multiplexing of samples, and all fragments are enriched with PCR to get a final cDNA library. Samples were then sequenced on the Illumina NEXTSEQ 500 Mid Output System, and analyzed using Illumina's BaseSpace software (www.illumina.com).