Three independently passaged biological replicates of mESCs (~3.5 × 107 cells per replicate) were cultured in "2i + LIF" ES medium supplemented with 1µM flavopiridol (HY-10005-10mg, Biotrend) to block the transcription. Cells were harvested at 0, 30, 60, 120 and 180 min after addition of flavopiridol followed by cell fractionation as described above.
Obtained fractions were mixed with Trizol LS and RNA was isolated following the manufacturer instructions. One microgram of total RNA was used as an input for TruSeq Stranded mRNA Library Prep Kit according to the instructions of the manufacturer. The multiplexed libraries were sequenced using HiSeq 4000 for pair-end 75 cycles by the BIH Genomics platform at the Max Delbrück Center for Molecular Medicine.
Obtained fractions were mixed with Trizol LS and RNA was isolated following the manufacturer instructions. One microgram of total RNA was used as an input for TruSeq Stranded mRNA Library Prep Kit according to the instructions of the manufacturer. The multiplexed libraries were sequenced using HiSeq 4000 for pair-end 75 cycles by the BIH Genomics platform at the Max Delbrück Center for Molecular Medicine.
