NextSeq 500 paired end sequencing; RNA-seq of human embryonic stem cells during cell cycle progression
RNA-seq of human embryonic stem cells during cell cycle progression
FUCCI hESCs were washed with PBS and collected by incubating with Cell Dissociation Buffer (Gibco) for 10 min at 37°C. Cells were washed with 1% PBS/BSA and incubated in 1% PBS/BSA with anti-TRA-1-60-antibody mouse monoclonal IgM (1:100, Santa Cruz Biotechnology, Inc.) for 20 min on ice, together with the secondary antibody, Alexa Fluor® 647 goat anti-mouse IgM (1:1,000, Thermo Fisher). Cells were washed with PBS, re-suspended in 6ml of PBS and sorted into the different cell cycle phases using BD FACS AriaTM III, BD Fusion or BD Influx sorters by the Cambridge NIHR BRC Cell Phenotyping Hub. RNA was extracted using GenEluteTM Mammalian Total RNA Miniprep Kit and On-Column DNase I Digestion Set (Sigma Aldrich) according to manufacturer instructions. Polyadenylated (poly-A) purified opposing strand-specific mRNA library libraries were prepared from 1μg of total RNA using the TruSeq Stranded mRNA sample preparation kit (Illumina).