Illumina HiSeq 2500 paired end sequencing; RNA-seq of mouse embryonic stem cells with altered Zfp207 expression
RNA-seq of mouse embryonic stem cells with altered Zfp207 expression
CCE murine ESCs were cultured on 0.1% gelatin-coated tissue culture plates under feeder free culture conditions at 37°C with 5% CO2 in a humidified incubator. The media composition consists of Dulbecco's modified Eagle's medium (DMEM) high glucose, 15% fetal bovine serum (FBS; Gibco), 1% MEM non-essential amino acids (Sigma-Aldrich), 0.1 mM 2-β mercaptoethanol, 1% L-glutamine (Hyclone) and 1% penicillin/streptomycin (Gibco) and Leukemia inhibitory factor (LIF; R&D systems). Early passage mouse ESCs were transducedwith the lentiviral particles in mouse ESC media supplemented with polybrene (8μg/ml) for 24 hours. After 36 hours, infected cells were treated with puromycin (2μg/ml)for additional 6 days. Extraction of total RNA was performed using RNeasy Plus Mini Kit (Qiagen). One μg of total RNA was used for complementary DNA synthesis using RevertAid First Strand cDNA Synthesis kit (Invitrogen) as per the manufacturer's instructions. RNAseq library preparation was carried out at Novogene facilities (https://en.novogene.com/) following Illumina guidelines.