Transcriptional profiling of LIF stimulation in mouse embryonic stem cells
Transcriptional profiling of LIF stimulation in mouse embryonic stem cells
Cells were cultured in 2i medium (N2B27, 3uM CHIR99021, 1uM PD0325901) in the presence of 100 U/ml murine leukemia inhibitory factor (LIF) for 24 h. RNA was extracted with the TRIzol method followed by DNase treatment. Ribosomal RNA was depleted using RiboMinus (Invitrogen), and rRNA-depleted samples were sheared by ultrasonication on a Covaris S2 for 90s with the following parameter settings: Duty Cycle = 10, Cycles per Burst = 200, Intensity = 5. Fragmented RNA was reverse-transcribed with random hexamer primers using SuperScript III (Invitrogen) at 50C for 2h in the presence of 6 ug/ml actinomycin D to inhibit the generation of second-strand products. Second-strand cDNA was synthesized by DNA Polymerase I for 2h at 16C in the presence of RNase H and with dUTPs substituted for dTTPs. End repair of double-strand cDNAs was carried out with T4 DNA polymerase and T4 polynucleotide kinase (New England Biolabs). Blunt-end, 3prime-phosphorylated products were 3prime-adenylated by exo- Klenow fragment in the presence of dATPs and ligated to sequencing adapters (Illumina) by T4 DNA ligase (New England Biolabs) at 20C for 30 minutes. Following adapter ligation, the second strand of the library constructs was digested with uracil DNA glycosylase (UDG) and apurinic/apyrimidinic endonuclease 1 (APE 1) for 30min at 37C. PCR amplification of first-strand library constructs was carried out with Phusion DNA polymerase (Finnzymes) for 15 cycles. Purification of reaction products between each step was performed with Ampure XP paramagnetic beads (Beckman Coulter).